Artificially-Manipulated Neovascularization Regulatory System

ABSTRACT

The present invention relates to an artificially manipulated neovascularization-associated factor for regulating neovascularization and a use thereof. More particularly, the present invention relates to a system for artificially regulating neovascularization, which includes an artificially manipulated neovascularization-associated factor for regulating neovascularization and/or a composition for artificially manipulating the neovascularization-associated factor. In a specific aspect, a neovascularization regulatory system including a neovascularization-associated factor such as artificially manipulated VEGFA, HIF1A, ANGPT2, EPAS1, or ANGPTL4 and/or an expression product thereof is provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of International Patent Application No. PCT/KR2017/009078, filed 21 Aug. 2017, which claims the priority and the benefit of U.S. Provisional Patent Application No. 62/376,998, filed on Aug. 19, 2016, the disclosures of which are incorporated herein by reference in their entirety.

FIELD

The present invention relates to an artificially manipulated neovascularization-associated factor for regulating neovascularization and a use thereof. More particularly, the present invention relates to a system capable of artificially regulating neovascularization, which includes an artificially manipulated neovascularization-associated factor for regulating neovascularization and/or a composition able to be used in artificial manipulation of the neovascularization-associated factor.

BACKGROUND

Excessive neovascularization found in many cases of severe diseases occurs in diseases such as cancer, macular degeneration, diabetic retinopathy, arthritis and psoriasis. In such a state, new blood vessels are provided to tissue with a disease, resulting in destroyed normal tissue, and in the case of cancer, new blood vessels allow tumor cells to enter the circulation system and thus settle in another organ (tumor metastasis).

Particularly, since cancer cells receive nutrients through neovascularization and are metastasized to another organ, neovascularization is essential for growth and metastasis of cancer. It has been known that there is an actual close relationship between the density of capillaries generated in cancer tissue and probability of cancer metastasis in various types of cancer. In addition, rheumatoid arthritis, which is the representative disease among inflammatory diseases, is caused by an autoimmune disorder, however, during the development of the disease, chronic inflammation generated in the synovial cavity between joints leads to neovascularization, resulting in destroyed cartilage. Various ophthalmologic diseases leading to blindness in several million people in the world every year are also caused by neovascularization. As a representative example, diabetic blindness is a diabetic complication, and refers to an invasion of capillaries generated in the retina to the vitreous body through neovascularization, ending up in blindness. Therefore, neovascularization inhibitory substances may be usefully employed as therapeutic agents and preventive agents for various diseases such as cancer, rheumatoid arthritis and diabetic blindness, in which continuous neovascularization occurs.

Meanwhile, conventionally, inhibition of signaling of vascular endothelial growth factors (VEGFs) in order to inhibit neovascularization had been actively studied. However, in the conventional art, initially, neovascularization seemed to be inhibited, and then there was a side effect in which cancer cells became more aggressive because the pathway of an anticancer agent to the cancer cells was also inhibited.

As such, while a variety of studies to treat diseases induced by neovascularization are progressing, there is almost no fundamental method for treating such a disease.

Particularly, there is no method for treating a severe disease such as cancer or cancer metastasis caused by neovascularization, blindness caused by retinal or corneal degeneration, and therefore, there is an urgent demand for developing such a fundamental method for treating a neovascularization-associated disease.

SUMMARY Technical Problem

To solve the above problems, the present invention relates to an artificially manipulated neovascularization system, which has an improved neovascularizing effect. More particularly, the present invention relates to an artificially manipulated neovascularization-associated factor and a neovascularization system which has a function artificially modified by the factor.

The present invention also relates to a neovascularization-associated factor genetically manipulated or modified for a specific purpose.

As an exemplary embodiment, the present invention is directed to providing an artificially manipulated neovascularization-regulating system.

As an exemplary embodiment, the present invention is directed to providing an artificially manipulated neovascularization-associated factor and an expression product thereof.

As an exemplary embodiment, the present invention is directed to providing a composition for manipulating a gene to manipulate a neovascularization-associated factor and a method for utilizing the same.

As an exemplary embodiment, the present invention is directed to providing a method for regulating neovascularization.

As an exemplary embodiment, the present invention is directed to providing a pharmaceutical composition for treating a neovascularization-associated disease and various uses thereof.

As an exemplary embodiment, the present invention is directed to providing an artificially manipulated neovascularization-associated factor, for example, VEGFA, HIF1A, ANGPT2, EPAS1, ANGPTL4, etc., and/or expression products thereof.

As an exemplary embodiment, the present invention is directed to providing a composition for manipulating a gene to enable artificial manipulation of a neovascularization-associated factor, for example, VEGFA, HIF1A, ANGPT2, EPAS1, ANGPTL4, etc.

As an exemplary embodiment, the present invention is directed to providing a therapeutic use of an artificially manipulated neovascularization-associated factor, for example, VEGFA, HIF1A, ANGPT2, EPAS1, ANGPTL4, etc., and/or a composition for manipulating a gene to enable the artificial manipulation.

As an exemplary embodiment, the present invention is directed to providing an additional use of an artificially manipulated neovascularization-associated factor, for example, VEGFA, HIF1A, ANGPT2, EPAS1, ANGPTL4, etc., and/or a composition for manipulating a gene to enable the artificial manipulation.

Technical Solution

To solve these problems, the present invention relates to a system for artificially regulating neovascularization, which includes an artificially manipulated neovascularization-associated factor for regulating neovascularization and/or a composition for artificially manipulating the neovascularization-associated factor.

The present invention provides an artificially manipulated neovascularization-associated factor for a specific purpose.

The term “neovascularization-associated factor” encompasses a variety of non-natural, artificially manipulated substances capable of having a neovascularization regulating function, which directly participate in or indirectly affect neovascularization. The substances may be DNA, RNA, genes, peptides, polypeptides or proteins. For example, the substances may be genetically manipulated or modified genes or proteins expressed in an immune cells. The neovascularization-associated factor may promote or increase neovascularization, or conversely, suppress or inhibit neovascularization.

In addition, it may induce, activate or inactivate a neovascularization environment or a neovascularization-inhibiting environment.

In an exemplary embodiment of the present invention, the neovascularization-associated factor may be, for example, an artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene or ANGPTL4 gene.

In an exemplary embodiment of the present invention, the neovascularization-associated factor may include two or more artificially manipulated genes. For example, two or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene may be artificially manipulated.

Therefore, in an exemplary embodiment of the present invention, one or more artificially manipulated neovascularization-associated factors selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, which have undergone modification in a nucleic acid sequence, are provided.

The modification in a nucleic acid sequence may be non-limitedly, artificially manipulated by a guide nucleic acid-editor protein complex.

The term “guide nucleic acid-editor protein complex” refers to a complex formed through the interaction between a guide nucleic acid and an editor protein, and the nucleic acid-protein complex includes the guide nucleic acid and the editor protein.

The guide nucleic acid-editor protein complex may serve to modify a subject. The subject may be a target nucleic acid, a gene, a chromosome or a protein.

For example, the gene may be a neovascularization-associated factor, artificially manipulated by a guide nucleic acid-editor protein complex, wherein the neovascularization-associated factor artificially manipulated includes one or more modifications of nucleic acids which is at least one of a deletion or insertion of one or more nucleotides, a substitution with one or more nucleotides different from a wild-type gene, and an insertion of one or more foreign nucleotide, in a proto-spacer-adjacent motif (PAM) sequence in a nucleic acid sequence constituting the neovascularization-associated factor or in a continuous 1 bp to 50 bp the base sequence region adjacent to the 5′ end and/or 3′ end thereof, or a chemical modification of one or more nucleotides in a nucleic acid sequence constituting the neovascularization-associated factor.

The modification of nucleic acids may occur in a promoter region of the gene.

The modification of nucleic acids may occur in an exon region of the gene. In one exemplary embodiment, 50% of the modifications may occur in the upstream section of the coding regions of the gene.

The modification of nucleic acids may occur in an intron region of the gene.

The modification of nucleic acids may occur in an enhancer region of the gene.

The PAM sequence may be, for example, one or more of the following sequences (described in the 5′ to 3′ direction):

-   -   NGG (N is A, T, C or G);     -   NNNNRYAC (each of N is independently A, T, C or G, R is A or G,         and Y is C or T);     -   NNAGAAW (each of N is independently A, T, C or G, and W is A or         T);     -   NNNNGATT (each of N is independently A, T, C or G);     -   NNGRR(T) (each of N is independently A, T, C or G, R is A or         G,); and     -   TTN (N is A, T, C or G).

The editor protein may be derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina.

In one exemplary embodiment, the editor protein may be one or more selected from the group consisting of a Streptococcus pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived Cas9 protein, a Streptococcus aureus-derived Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a Cpf1 protein. As an example, the editor protein may be a Streptococcus pyogenes-derived Cas9 protein or a Campylobacter jejuni-derived Cas9 protein.

In addition, in another embodiment, the present invention provides a guide nucleic acid, which is capable of forming a complementary bond with respect to target sequences of SEQ ID NOs: 1 to 1522, for example, SEQ ID Nos: 1 to 79 in the nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, respectively.

The guide nucleic acid may form a complementary bond with a part of nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene. It may create 0 to 5, 0 to 4, 0 to 3, or 0 to 2 mismatches. As an exemplary example, the guide nucleic acid may be nucleotides forming a complementary bond with one or more of the target sequences of SEQ ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79, respectively.

For example, the present invention may provide one or more guide nucleic acids selected from the group as described below:

-   -   a guide nucleic acid capable of forming a complementary bond         with respect to the target sequences of SEQ ID NOs: 3, 4, 7, 9,         10 and 11 in the nucleic acid sequence of the VEGFA gene,         respectively;     -   a guide nucleic acid capable of forming a complementary bond         with respect to the target sequences of SEQ ID NOs: 14, 18, 19,         20, 26, 29 and 31 in the nucleic acid sequence of the HIF1A         gene, respectively;     -   a guide nucleic acid capable of forming a complementary bond         with respect to the target sequences of SEQ ID NOs: 33, 34, 37,         38, 39 and 43 in the nucleic acid sequence of the ANGPT2 gene,         respectively;     -   a guide nucleic acid capable of forming a complementary bond         with respect to the target sequences of SEQ ID NOs: 47, 48, 49,         50, 53, 54 and 55 in the nucleic acid sequence of the EPAS1         gene, respectively; and     -   a guide nucleic acid capable of forming a complementary bond         with respect to the target sequences of SEQ ID NOs: 64, 66, 67,         73, 76 and 79 in the nucleic acid sequence of the ANGPTL4 gene,         respectively.

The guide nucleic acid may be non-limitedly 18 to 25 bp, 18 to 24 bp, 18 to 23 bp, 19 to 23 bp, or 20 to 23 bp nucleotides.

In addition, the present invention provides a composition for gene manipulation, which may be employed in artificial manipulation of a neovascularization-associated factor for a specific purpose.

The composition for gene manipulation may include a guide nucleic acid-editor protein complex or a nucleic acid sequence encoding the same.

The composition for gene manipulation may include:

-   -   (a) a guide nucleic acid capable of forming a complementary bond         with respect to each of target sequences of SEQ ID NOs: 1 to         1522, for example, SEQ ID NOs: 1 to 79 in the nucleic acid         sequences of one or more genes selected from the group         consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an         EPAS1 gene and an ANGPTL4 gene, respectively or a nucleic acid         sequence encoding the guide nucleic acid;     -   (b) an editor protein including one or more proteins selected         from the group consisting of a Streptococcus pyogenes-derived         Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a         Streptococcus thermophilus-derived Cas9 protein, a Streptococcus         aureus-derived Cas9 protein, a Neisseria meningitidis-derived         Cas9 protein, and a Cpf1 protein or a nucleic acid sequence         encoding the same.

In one exemplary embodiment, the guide nucleic acid may be a nucleic acid sequence which forms a complementary bond with respect to one or more of the target sequences of SEQ ID NOs: 3, 4, 7, 9, 10 and 11 (VEGFA), SEQ ID NOs: 14, 18, 19, 20, 26, 29 and 31 (HIF1A), SEQ ID NOs: 33, 34, 37, 38, 39 and 43 (ANGPT2), SEQ ID NOs: 47, 48, 49, 50, 53, 54 and 55 (EPAS1), and SEQ ID NOs: 64, 66, 67, 73, 76 and 79 (ANGPTL4), respectively.

In one exemplary embodiment, the composition for gene manipulation may be a viral vector system.

The viral vector may include one or more selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a herpes simplex virus.

In an exemplary embodiment, the present invention provides a method for artificially manipulating cells, which includes: introducing

-   -   (a) a guide nucleic acid which is capable of forming a         complementary bond with respect to the target sequences of SEQ         ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79 in the         nucleic acid sequences of one or more genes selected from the         group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene,         an EPAS1 gene and an ANGPTL4 gene, respectively, or a nucleic         acid sequence encoding the same; and     -   (b) an editor protein including one or more proteins selected         from the group consisting of a Streptococcus pyogenes-derived         Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a         Streptococcus thermophilus-derived Cas9 protein, a Streptococcus         aureus-derived Cas9 protein, a Neisseria meningitidis-derived         Cas9 protein, and a Cpf1 protein, respectively, or a nucleic         acid sequence encoding the same to cells.

The guide nucleic acid and the editor protein may be present in one or more vectors in the form of a nucleic acid sequence, or may be present in a complex formed by coupling the guide nucleic acid with the editor protein.

The introduction may be performed in vivo or ex vivo.

The introduction may be performed by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles and a protein translocation domain (PTD) fusion protein method.

The viral vector may be one or more selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a herpes simplex virus.

In another exemplary embodiment, the present invention provides a pharmaceutical composition for treating a neovascularization-associated disease.

The pharmaceutical composition may include a composition for gene manipulation which may be employed in artificial manipulation of a neovascularization-associated factor.

The formulation of the composition for gene manipulation is the same as described above.

In an exemplary embodiment, the present invention provides a method for obtaining information about the sequences of target sites that are artificially manipulated from a subject by sequencing one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene.

In addition, the present invention provides a method for constructing libraries using the information obtained thereby.

In an exemplary embodiment, the present invention provides a kit for gene manipulation, which includes the following components:

-   -   (a) a guide nucleic acid capable of forming a complementary bond         with respect to the target sequences of SEQ ID NOs: 1 to 1522,         for example, SEQ ID NOs: 1 to 79 in the nucleic acid sequences         of one or more genes selected from the group consisting of a         VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an         ANGPTL4 gene, respectively, or a nucleic acid sequence encoding         the same; and     -   (b) an editor protein including one or more proteins selected         from the group consisting of a Streptococcus pyogenes-derived         Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a         Streptococcus thermophilus-derived Cas9 protein, a Streptococcus         aureus-derived Cas9 protein, a Neisseria meningitidis-derived         Cas9 protein, and a Cpf1 protein, respectively, or a nucleic         acid sequence encoding the same.

The gene of interest may be artificially manipulated using such a kit.

In one exemplary embodiment, the present invention may provide a composition for treating a neovascularization-related disorder, which includes:

-   -   a guide nucleic acid capable of forming a complementary bond         with one or more target sequences in the nucleic acid sequences         of one or more genes selected from the group consisting of a         VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an         ANGPTL4 gene, respectively, or a nucleic acid sequence encoding         the same; and an editor protein or a nucleic acid sequence         encoding the same.

The target sequences may be one or more sequences of SEQ ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79.

In one exemplary embodiment, a Campylobacter jejuni-derived Cas9 protein may be employed as the editor protein.

In one exemplary embodiment, the neovascularization-related disorder may be ischemic retinopathy or retinopathy of prematurity.

In one exemplary embodiment, the present invention provides all aspects of uses of an artificially manipulated neovascularization-associated factor or a composition for gene manipulation which is employed in artificial manipulation of the neovascularization-associated factor for treating a disease in a target.

Targets for treatment may be mammals including primates such as humans, monkeys, etc., rodents such as mice, rats, etc., and the like.

Advantageous Effects

An artificially manipulated neovascularization-associated factor and a neovascularization system whose function is artificially modified thereby can be effectively used to treat a neovascularization-related disease, for example, a neovascularization-related ocular disease. The efficiency of the neovascularization system can be improved by modulation of a variety of in vivo mechanisms in which various neovascularization-associated factors are involved.

For example, one or more genes of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene can be utilized.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1, 2, 3, 4, 5, 6, and 7 show the reducing effect on a laser-induced choroidal neovascularization (CNV) area due to CjCas9 targeting Vegfa or Hif1a in mouse models with age-related macular degeneration (AMD): FIG. 1 CjCas9 target sequences in Vegfa and Hif1a/HIF1A genes (the PAM sequence and the target sequence of sgRNA are marked with a dotted line and solid line, respectively), FIG. 2 the all-in-one AAV vector encoding CjCas9 and an in vivo test schedule, FIG. 3 graphs of indel frequencies at Rosa26, Vegfa, and Hif1a target sites in RPE cells (Error bars=s.e.m. (a control not injected with AAV: n=4, AAV-CjCa9-injected test group: n=5), Student's t-tests, * p<0.05, *** p<0.001), FIG. 4a graph of Vegfa expression levels measured by ELISA in RPE cells (Error bars=s.e.m. (a control not injected with AAV: n=4, AAV-CjCa9-injected test group: n=5), One-way ANOVA and Tukey post-hoc tests, * p<0.05, *** p<0.001), FIG. 5a graph of indel frequencies at off-target sites (the mismatched base sequence is marked with a solid line, and the PAM sequence is marked with a dotted line), FIG. 6 laser-induced CNV areas of eyeballs of mice injected with AAV9-CjCa9 targeting Rosa26, Vegfa, or Hif1a, stained with isolectin B4 (Scale bar=200 μm), and FIG. 7 a graph of the laser-induced CNV areas (%) (Error bars=s.e.m.(n=17-18), One-way ANOVA and Tukey post-hoc tests, * p<0.05, ** p<0.01, *** p<0.001, ns: not significant).

FIG. 8 shows images of in vivo expression of eGFP with CjCas9 in mouse RPE cells (n=6, anti-GFP antibody (green) and DAPI (blue), Scale bar=20 μm).

FIGS. 9 and 10 show opsin-positive areas in the retinas of AAV/CjCas9-injected mice: FIG. 9 images of opsin-positive areas corresponding to RPE cells expressing HA-tagged CjCas9 in Rosa26-, Vegfa-, or Hif1a-specific CjCas9-injected mice (Opsin: red, HA: green, and DAPI: blue, Scale bar=20 μm, ONL: outer nuclear layer, IS: inner segment of photoreceptor cells, OS: outer segment of photoreceptor cells); and FIG. 10 a graph of relative opsin-positive areas (%) (Error bars=s.e.m.(n=4), One-way ANOVA and Tukey post-hoc tests, * p<0.05).

FIGS. 11 and 12 show reduced expression of VEGF due to CjCas9 targeting Vegfa or Hif1a in retinal tissue: FIG. 11a graph of indel frequencies at target sites of Rosa26, Vegfa and Hif1a in retinal cells (Error bars=s.e.m. (a control not injected with AAV: n=4, AAV-CjCa9-injected test group: n=5), Student's t-tests, * p<0.05, ** p<0.01, *** p<0.001), FIG. 12a graph of Vegfa expression levels in retinal cells, measured using ELISA (Error bars=s.e.m. (n=6-7), One-way ANOVA and Tukey post-hoc tests, * p<0.05, *** p<0.001).

FIG. 13 shows the reducing effect of CjCas9 targeting Vegfa on vascular leakage and blood leakage in retinas of diabetic retinopathy mouse models, (a) an in vivo test schedule, (b) images of reduced effects on vascular leakage and blood leakage in mouse retinas injected with AAV2-CjCa9 targeting Vegfa.

FIGS. 14 and 15 show the reducing effect of CjCas9 targeting Vegfa on vascular leakage and blood leakage in retinas of diabetic retinopathy mouse models: FIG. 14 is an in vivo test schedule (a), images of the reducing effect on vascular leakage and blood leakage in mouse retinas injected with AAV2-CjCa9 targeting Vegfa (b), and FIG. 15 is a graph of relative vascular leakage (%) due to CjCas9 targeting Rosa26 or Vegfa.

FIG. 16 shows CjCas9 target site screening results and indel frequencies of human VEGFAs for gene manipulation.

FIGS. 17 and 18 show the result of CjCas9 target site screening of human HIF1As for gene manipulation: FIG. 17 CjCas9 target site screening results and indel frequencies of human HIF1As, and FIG. 18 target sites of HIF1As, conserved between various mammals.

FIG. 19 shows CjCas9 target site screening results and indel frequencies of human ANGPT2s for gene manipulation.

FIG. 20 shows CjCas9 target site screening results and indel frequencies of human EPAS1s for gene manipulation.

FIG. 21 shows CjCas9 target site screening results and indel frequencies of human ANGPTL4s for gene manipulation.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or identical to those described herein can be used in practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, materials, methods and examples are merely illustrative, and not intended to be limitive.

One aspect of the present invention relates to an artificially manipulated neovascularization system, which has a regulated neovascularization effect.

Specifically, the present invention relates to combination of various aspects capable of regulating neovascularization or improving or treating a neovascularization-associated disease by artificially manipulating a neovascularization-associated factor. The present invention includes a neovascularization-associated factor whose function is artificially modified, a method of manufacturing the same, a composition including the same, and a therapeutic use thereof.

Another aspect of the present invention relates to an additional regulating system with a third in vivo mechanism, concomitant with various functions of a specific factor (e.g., a gene known as a neovascularization-associated factor, etc.) whose function is artificially modified.

Specifically, targeting of a third in vivo function as well as a neovascularization function in which artificially manipulated specific factors are involved may lead to the regulation of corresponding mechanisms. The present invention includes a neovascularization-associated factor whose function is artificially modified, a method for manufacturing the same, a composition including the same, and therapeutic uses thereof for improving or treating a disease associated with the third function.

Neovascularization

An exemplary embodiment of the present invention relates to improvement and modification of a neovascularization-associated system.

The term “neovascularization” refers to a process of tissue vascularization, including generation, development and/or differentiation of new blood vessels. Here, neovascularization encompasses angiogenesis and vasculogenesis. Neovascularization may be closely related to various factors to promote or inhibit proliferation of vascular endothelial cells.

Neovascularization encompasses all mechanisms for extension from existing blood vessels, new generation of blood vessels from precursor cells, and/or an increase in diameter of an existing small blood vessel.

In addition, neovascularization encompasses all mechanisms associated with formation of new vessels, which is involved in vascular leakage or repair of damaged blood vessels.

The vascularization includes mechanisms concomitant with excessive and/or abnormal neovascularization in various severe disease states.

For example, in diseases such as cancer, macular degeneration, diabetic retinopathy, arthritis and psoriasis, excessive neovascularization occurs. In such a disease state, new blood vessels are provided to tissue with a disease, and normal tissue is damaged. In the case of cancer, new blood vessels allow tumor cells to enter into the circulation system and thus enable them to settle in another organ (tumor metastasis).

In one exemplary embodiment, the neovascularization may be ocular vascularization.

For example, the neovascularization may be found in eye diseases such as AMD, diabetic retinopathy and the like. Particularly, AMD is the most common cause of legal irreversible blindness in older people over the age of 65 in the US, Canada, England, Wales, Scotland and Australia, and about 10% to 15% of the patients show exudative (wet) diseases. The exudative AMD is characterized by neovascularization and disease-causing angiogenesis.

For example, ocular neovascularization may include choroidal neovascularization (CNV), corneal neovascularization and/or rubeosis iridis.

CNV is the vascularization in the choroid layer, and rapidly occurs in people having a defect in the Bruch's membrane, which is the innermost layer of the choroid. In addition, CNV is associated with an excessive amount of vascular endothelial growth factor (VEGF). CNV may cause excessive myopia, malignant myopia, or neovascular degenerative macular degeneration (e.g., wet macular degeneration).

Corneal neovascularization is the growth of new blood vessels from the pericorneal plexus in the periphery of the cornea into avascular corneal tissue due to oxygen deprivation, and may be mainly caused by congenital or inflammatory (e.g., rejection after corneal transplantation, grafted tissue or host diseases, atopic conjunctivitis, injections, ocular pemphigoid, Lyell's syndrome, and Stevens-Johnson syndrome), infectious (e.g., bacterial (chlamydia, syphilis, pseduomonas), viral (herpes simplex virus and herpes zoster virus), fungal (candida, aspergillus, fusarium), parasistic (onchocerca volvolus), degenerative, traumatic and iatrogenic (e.g., the wearing of contact lenses) diseases.

Rubeosis iridis is the vascularization on the surface of the iris, associated with diabetic retinopathy, and also known to be caused by central retinal vein occlusion, ocular ischemic syndrome, chronic retinal detachment, and the like.

In a certain embodiment, the neovascularization may be associated with survival, proliferation, persistency, cytotoxicity, and a cytokine-release function of vascular endothelial cells.

In a certain embodiment, neovascularization may be associated with an increase in the expression of an angiogenic cytokine.

In a certain embodiment, the neovascularization may be associated with functions of a receptor of vascular endothelial cells.

In a certain embodiment, the neovascularization may be associated with a migration ability of vascular endothelial cells.

In a certain embodiment, the neovascularization may be associated with an attachment ability of vascular endothelial cells.

Neovascularization-Associated Factor

Neovascularization-Associated Factor

One embodiment of the present invention relates to an artificially manipulated or modified neovascularization-associated factor.

The term “neovascularization-associated factor” includes all elements directly participating in or indirectly affecting vasculogenesis or angiogenesis. Here, the elements may include DNA, RNA, genes, peptides, polypeptides or proteins.

In an exemplary embodiment, the neovascularization-associated factor may include various substances which can have a non-natural, that is, artificially manipulated, neovascularization regulating function. For example, it may be a genetically manipulated or modified genes or proteins expressed in an immune cells.

The term “artificially manipulated” means an artificially modified state, which is not a naturally occurring state.

The term “genetically manipulated” means that a genetic modification is artificially introduced to biological or non-biological substances cited in the present invention, and may be, for example, genes and/or gene products (polypeptides, proteins, etc.) in which their genomes are artificially modified for a specific purpose.

As an exemplary example, the present invention provides a neovascularization-associated factor which is genetically manipulated or modified for a specific purpose.

Genes or proteins having the functions listed below may have multiple types of functions, not only one type of neovascularization-associated function. In addition, as needed, two or more neovascularization functions and factors may be provided.

The neovascularization-associated factor may promote or increase neovascularization.

The neovascularization-associated factor may suppress or inhibit neovascularization.

The neovascularization-associated factor may induce or activate a neovascularization environment.

The neovascularization-associated factor may induce a neovascularization-inhibited environment or inactivate a neovascularization environment.

The neovascularization-associated factor may regulate (promote, increase, suppress and/or inhibit etc.) neovascularization.

The neovascularization-associated factor may be utilized in improvement and treatment of a neovascularization-associated disease.

In an exemplary embodiment, the neovascularization-associated factor may be one or more selected from the group consisting of ABCA1, ACAT, ACC2, ADAMTS12, ADCY2, ADIPOQ, ADIPOR1, ADIPOR2, ADRB2, AGPAT5, AIP4, AKAP2, AKR1C2, AMPK, ANG2, ANGPT2, ANGPTL4, ANK1, ANXA1, APOA1, ARHGAP17, ATP10A, AUH, AUTOTAXIN, BAI3, BCAR1, BIN1, BMP3A, CA10, CAMK1D, CAMKK2, CD36, CD44, CDC42, CDH13, CHAT, CNTFR, COL4A2, CPT, CSH1, CTNN, CUBN, CYP7B1, CYSLTR1, CYSLTR2, DGKB, DGKH, DGKZ, DHCR7, DHFR, DRD2, DRD5, EDG1, EDG2, EDG3, EDG4, EDG5, EDGE, EDG7, EDGE, EDNRA, EHHADH, ENPP6, EPAS1, ERBB4, ERK1, ERK2, ESRRG, ETFA, F2, FDPS, FGF2, FLNA, FLT4, FOXO1, FOXO3A, FTO, GABBR2, GATA3, GH1, GNA12, GNA13, GRK2, GRK5, GRM5, HAPLN1, HAS1, HAS2, HAS3, HCRTR2, HIF1A, HSD11B1, HYAL1, HYAL2, HYAL3, IL20RA, IL20RB, IL6ST, IL8, ITGA6, ITGB1, KDR, LAMA1, LDLR, LEPR, LEPTIN, LIFR, LIPL2, LKB1, LRP, LTBP2, MAT2B, ME1, MEGALIN, MERLIN, MET, MGST2, MMP2, MMP9, MTOR, MTR, NCK2, NEDD9, NFKB1, NFKBIB, NOS2A, NOS3, NR112, NR3C2, NRG1, NRP1, NRP2, OPRS1, OSBPL10, OSBPL3, OSTEOPONTIN, P2RY1, P2RY12, PAI1, PAI2, PAK1, PAK6, PALLD, PAP1, PAR1, PAXILLIN, PC, PCTP, PDE11A, PDE1A, PDE3A, PDE4D, PDE5, PDGFA, PDGFB, PDGFRA, PDGFRB, PI3K, PITPNC1, PKA, PKCD, PLA1A, PLA2, PLAT, PLAU, PLCB1, PLD1, PLD2, PLG, PLXDC2, PPARA, PPARG, PPARGC1B, PRKG1, PRL, PTGS2, PTN, PTPN11, PYK2, RAC1, RAS, RHEB, RHOA, ROCK1, ROCK2, RPS6KA1, RPS6KB2, SCARB1, SCHIP1, SGPP2, SLC25A21, SMAD3, SMAD4, SNCA, SORBS2, SPLA2, SPOCK1, SRD5A1, SREBF1, SREBF2, STAT3, TGFBR1, TGFBR2, TGFBR3, THBS1, THBS2, THEM2, THRB, TIAM1, TIMP2, TLL2, TSC1, TSC2, TSPO, VEGFA, VEGFR1, and YES1.

As an exemplary example of the present invention, the neovascularization-associated factor may be one or more selected from the group consisting of VEGFA, HIF1A, ANGPT2, EPAS1, and ANGPTL4.

In a certain embodiment, the neovascularization-associated factor may be VEGFA.

A vascular endothelial growth factor A (VEGFA) gene is a gene (full-length DNA, cDNA or mRNA) encoding a VEGFA protein also called MVCD1, VEGF or VPF. In one example, the VEGFA gene may be one or more selected from the group consisting of the following genes, but the present invention is not limited thereto: genes encoding human VEGFA (e.g., NCBI Accession No. NP_001020537.2 or NP_001020538.2), for example, VEGFA genes represented by NCBI Accession No. NM_001025366.2, NM_001025367.2, NM_003376, or NG_008732.1.

In a certain embodiment, the neovascularization-associated factor may be HIF1A.

A hypoxia-inducible factor 1-alpha (HIF-1-alpha; HIF1A) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a HIF1A protein also called HIF1, MOP1, PASD8 or bHLHe78. In an example, the HIF1A gene may be one or more selected from the group consisting of the following genes, but the present invention is not limited thereto: genes encoding human HIF1A (e.g., NCBI Accession No. NP_001230013.1 or NP_001521.1), for example, HIF1A genes represented by NCBI Accession No. NM_001243084.1, NM_001530.3, NM_181054.2, or NG_029606.1.

In a certain embodiment, the neovascularization-associated factor may be ANGPT2.

An angiopoietin-2 (ANGPT2) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding an ANGPT2 protein also called AGPT2 or ANG2. In an example, the ANGPT2 gene may be one or more selected from the group consisting of the following genes, but the present invention is not limited thereto: genes encoding human ANGPT2 (e.g., NCBI Accession No. NP_001112359.1, NP_001112360.1 or NP_001138.1), for example, ANGPT2 genes represented by NCBI Accession No. NM_001118887.1, NM_001118888.1, NM_001147.2 or NG_029483.1.

In a certain embodiment, the neovascularization-associated factor may be EPAS1.

An endothelial PAS domain-containing protein 1 (EPAS1) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding an EPAS1 protein also called ECYT4, HIF2A, HLF, MOP2, PASD2 or bHLHe73. In an example, the EPAS1 gene may be one or more selected from the group consisting of the following genes, but the present invention is not limited thereto: genes encoding human EPAS1 (e.g., NCBI Accession No. NP_001421.2, etc.), for example, EPAS1 genes represented by NCBI Accession No. NM_001430.4 or NG_016000.1.

In a certain embodiment, the neovascularization-associated factor may be ANGPTL4.

An angiopoietin-like 4 (ANGPTL4) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding an ANGPTL4 protein also called ARP4, FIAF, HARP, HFARP, NL2, PGAR, TGQTL or UNQ171. In an example, the ANGPTL4 gene may be one or more selected from the group consisting of the following genes, but the present invention is not limited thereto: genes encoding human ANGPTL4 (e.g., NCBI Accession No. NP_001034756.1 or NP_647475.1), for example, ANGPTL4 genes represented by NCBI Accession No. NM_001039667.2, NM_139314.2, or NG_012169.1.

The neovascularization-associated factor may be derived from mammals including primates such as human, monkeys and the like, rodents such as rats, mice and the like, etc.

Information about the genes may be obtained from a known database such as GeneBank of the National Center for Biotechnology Information (NCBI).

In an exemplary embodiment of the present invention, the neovascularization-associated factor, for example, VEGFA, HIF1A, ANGPT2, EPAS1 or ANGPTL4, may be an artificially manipulated neovascularization-associated factor.

In a certain embodiment, the artificially manipulated neovascularization-associated factor may be genetically manipulated.

The gene manipulation or modification may be achieved by artificial insertion, deletion, substitution or inversion occurring in a partial or entire region of the genomic sequence of a wild type gene. In addition, the gene manipulation or modification may be achieved by fusion of manipulation or modification of two or more genes.

For example, the gene is inactivated by such gene manipulation or modification, such that a protein encoded from the gene may not be expressed in the form of a protein having an innate function.

For example, the gene may be further activated by such gene manipulation or modification, such that a protein encoded from the gene is to be expressed in the form of a protein having an improved function, compared to the innate function. In an example, when a function of the protein encoded by a specific gene is A, a function of a protein expressed by a manipulated gene may be totally different from A or may have an additional function (A+B) including A.

For example, a fusion of two or more proteins may be expressed using two or more genes having different or complementary functions due to such gene manipulation or modification.

For example, two or more proteins may be expressed separately or independently in cells by using two or more genes having different or complementary functions due to such gene manipulation or modification.

The manipulated neovascularization-associated factor may promote or increase neovascularization.

The manipulated neovascularization-associated factor may suppress or inhibit neovascularization.

The manipulated neovascularization-associated factor may induce or activate a neovascularization environment.

The manipulated neovascularization-associated factor may induce a neovascularization inhibiting environment or inactivate a neovascularization environment.

The manipulated neovascularization-associated factor may regulate (promote, increase, suppress and/or inhibit) neovascularization.

The manipulated neovascularization-associated factor may be utilized in improvement and treatment of a neovascularization-associated disease.

The manipulation includes all types of structural or functional modifications of the neovascularization-associated factor.

The structural modification of the neovascularization-associated factor includes all types of modifications, which are not the same as those of a wild type existing in a natural state.

For example, when the neovascularization-associated factor is DNA, RNA or a gene, the structural modification may be the loss of one or more nucleotides.

The structural modification may be the insertion of one or more nucleotides.

Here, the inserted nucleotides include all of a subject including a neovascularization-associated factor and nucleotides entering from the outside of the subject.

The structural modification may be the substitution of one or more nucleotides.

The structural modification may include the chemical modification of one or more nucleotides.

Here, the chemical modification includes all of the addition, removal and substitution of chemical functional groups.

As another example, when the neovascularization-associated factor is a peptide, a polypeptide or a protein, the structural modification may be the loss of one or more amino acids.

The structural modification may be the insertion of one or more amino acids.

Here, the inserted amino acids include all of a subject including a neovascularization-associated factor and amino acids entering from the outside of the subject.

The structural modification may be the substitution of one or more amino acids.

The structural modification may include the chemical modification of one or more amino acids.

Here, the chemical modification includes all of the addition, removal and substitution of chemical functional groups.

The structural modification may be the partial or entire attachment of a different peptide, polypeptide or protein.

Here, the different peptide, polypeptide or protein may be a neovascularization-associated factor, or a peptide, polypeptide or protein having a different function.

The functional modification of the neovascularization-associated factor may include all types having an improved or reduced function, compared to that of a wild type existing in a natural state, and having a third different function.

For example, when the neovascularization-associated factor is a peptide, polypeptide or protein, the functional modification may be a mutation of the neovascularization-associated factor.

Here, the mutation may be a mutation that enhances or suppresses a function of the neovascularization-associated factor.

The functional modification may have an additional function of the neovascularization-associated factor.

Here, the additional function may be the same or a different function. In addition, the neovascularization-associated factor having the additional function may be fused with a different peptide, polypeptide or protein.

The functional modification may be the enhancement in functionality due to increased expression of the neovascularization-associated factor.

The functional modification may be the degradation in functionality due to decreased expression of the neovascularization-associated factor.

In an exemplary embodiment, the manipulated neovascularization-associated factor may be induced by one or more of the following mutations:

-   -   all or partial deletions of the neovascularization-associated         factor, that is, a gene to be manipulated (hereinafter, referred         to as a target gene), for example, deletion of 1 bp or longer         nucleotides, for example, 1 to 30, 1 to 27, 1 to 25, 1 to 23, 1         to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide of the         target gene,     -   substitution of 1 bp or longer nucleotides, for example, 1 to         30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to         5, 1 to 3, or 1 nucleotide of the target gene with a nucleotide         different from a wild type, and     -   insertion of one or more nucleotides, for example, 1 to 30, 1 to         27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3,         or 1 nucleotide (each independently selected from A, T, C and G)         into a certain position of the target gene.

A part of the modified target gene (“target region”) may be a continuous 1 bp or more, 3 bp or more, 5 bp or more, 7 bp or more, 10 bp or more, 12 bp or more, 15 bp or more, 17 bp or more, or 20 bp or more, for example, 1 bp to 30 bp, 3 bp to 30 bp, 5 bp to 30 bp, 7 bp to 30 bp, 10 bp to 30 bp, 12 bp to 30 bp, 15 bp to 30 bp, 17 bp to 30 bp, 20 bp to 30 bp, 1 bp to 27 bp, 3 bp to 27 bp, 5 bp to 27 bp, 7 bp to 27 bp, 10 bp to 27 bp, 12 bp to 27 bp, 15 bp to 27 bp, 17 bp to 27 bp, 20 bp to 27 bp, 1 bp to 25 bp, 3 bp to 25 bp, 5 bp to 25 bp, 7 bp to 25 bp, 10 bp to 25 bp, 12 bp to 25 bp, 15 bp to 25 bp, 17 bp to 25 bp, 20 bp to 25 bp, 1 bp to 23 bp, 3 bp to 23 bp, 5 bp to 23 bp, 7 bp to 23 bp, 10 bp to 23 bp, 12 bp to 23 bp, 15 bp to 23 bp, 17 bp to 23 bp, 20 bp to 23 bp, 1 bp to 20 bp, 3 bp to 20 bp, 5 bp to 20 bp, 7 bp to 20 bp, 10 bp to 20 bp, 12 bp to 20 bp, 15 bp to 20 bp, 17 bp to 20 bp, 21 bp to 25 bp, 18 bp to 22 bp, or 21 bp to 23 bp region of the base sequence of the gene.

Meanwhile, a different embodiment of the present invention relates to an additional system for regulating a third in vivo mechanism, concomitant with various functions of the above-described neovascularization-associated factors whose functions are artificially modified.

In one exemplary embodiment, VEGF may be involved in regulation of the third in vivo mechanism.

Since an increase in vascular permeability by VEGF may be a cause of edema, as well as tumor growth, artificially manipulated VEGF may increase a survival rate in various types of tumors (e.g., brain tumor, uterine cancer, vestibular schwannomas, etc.) or recover hearing loss, for example, by manipulation to inactivate VEGF. In addition, a decrease in vascular permeability by artificially manipulated VEGF may impart therapeutic effects on renal failure, arthritis, psoriasis, coronary disease, etc.

In addition, the artificially manipulated VEGF may impart a therapeutic effect on an autoimmune disease. For example, inflammation-inducing activity may be artificially regulated by VEGF, thereby imparting therapeutic effects on uveitis, rheumatoid arthritis, systemic lupus erythematosus, an inflammatory bowel disease, psoriasis, systemic sclerosis, multiple sclerosis, etc.

In addition, the artificially manipulated VEGF may impart a therapeutic effect on a mental disease. For example, a therapeutic effect on depression may be imparted by artificially regulating the expression of a neurotransmission-associated factor by VEGF.

In another embodiment, the artificially manipulated VEGF may be involved in the regulation of a third in vivo mechanism of HIF. The HIF may be HIF1 or HIF2.

The artificially manipulated HIF may regulate inflammation-inducing activity, thereby imparting therapeutic effects on uveitis, rheumatoid arthritis, systemic lupus erythematosus, an inflammatory bowel disease, psoriasis, systemic sclerosis, multiple sclerosis, etc.

In addition, the artificially manipulated HIF may provide a therapeutic effect on an autoimmune disease.

Likewise, the illustrative factors of the present invention which are artificially manipulated may regulate corresponding mechanisms by targeting the third in vivo function as well as the neovascularization function. One exemplary embodiment of the present invention includes such a neovascularization factor whose function is artificially modified and a method for manufacturing the same, a composition including the same, and uses of the factor and the composition for improving or treating a disease associated with a third function.

Neovascularization System

Neovascularization-Regulating System

In one aspect of the present invention, a neovascularization-regulating system for regulating neovascularization by artificially manipulating a neovascularization-associated factor is provided.

The term “neovascularization-regulating system” used herein includes all phenomena affecting the promotion, increase, suppression and/or inhibition of neovascularization by change of a function of an artificially manipulated neovascularization-associated factor, and also includes all substances, compositions, methods, and uses which are directly or indirectly involved in such a neovascularization-regulating system.

Each factor constituting such a neovascularization-regulating system is also generally called “neovascularization-regulating factor.”

The system of the present invention includes a modified in vivo mechanism, associated with an artificially manipulated neovascularization-associated factor.

In a certain embodiment, the expression of hematopoietic stem cell surface antigens such as CD34, CD117, CD133, etc. and vascular endothelial cell antigens such as Flk-1/KDR, Tie-2, etc. may be regulated by the artificially manipulated neovascularization-associated factor.

In a certain embodiment, angiogenesis in which new vessels are formed by sprouting and the growth of cells constituting a blood vessel may be regulated.

In a certain embodiment, the activity of direct angiogenic factors (DAFs) directly stimulating endothelial cells may be regulated. The growth and/or migration of endothelial cells may be promoted or inhibited.

For example, the DAFs may include vascular endothelial growth factors (VEGFs), basic fibroblast growth factors (bFGFs), hepatocyte growth factors (HGFs), epidermal growth factors (EGFs), thymidine phosphorylase (PD-ECGF), placental growth factors (PIGFs), transforming growth factors (TGFs), proliferin, interleukin-8 of a cytokine, angiogenin (angiogenesis-inducing protein), fibrin, nicotinamide (vitamin B complex), angiopoietin (angiogenesis-promoting protein), platelet activating factors (PAFs), 12-hydroxy eicosatetraenoate (12-HETE; a toxic degradation product of arachidonic acid, which is an angiogenesis-promoting factor of epithelial cells), matrix metalloproteases (MMPs), sphingosine 1-phosphate (S1P), and leptin.

In a certain embodiment, two different intercellular signaling pathways operating in blood vessel cells, that is, PDGF and VEGF signaling pathways may be utilized.

In a certain embodiment, the activity of indirect angiogenic factors (IAFs) inducing angiogenesis by formation of DAFs may be regulated by stimulating vascular pericytes.

In a certain embodiment, vascular endothelial cells may be differentiated from endothelial progenitor cells (EPCs), and thus a mechanism of forming a primary vascular plexus may be regulated.

In a certain embodiment, the degradability of extracellular matrix components for the migration of endothelial cells may be regulated.

In a certain embodiment, a cell migration-associated signaling pathway may be regulated.

In a certain embodiment, the activity of VEGF receptors such as VEGFR-1 (flt-1; fmslike-tyrosine kinase-1), VEGFR-2 (flk-1/KDR), and VEGFR-3, and a platelet derived growth factor (PDGF) receptor, or neuropilin-1 (NP-1) may be regulated.

In an exemplary embodiment, the neovascularization-regulating system includes a composition for manipulating a neovascularization-associated factor.

The composition for manipulation may be a composition capable of artificially manipulating a neovascularization-associated factor, and preferably, a composition for gene manipulation.

Hereinafter, the composition for gene manipulation will be described.

Composition for Manipulating Neovascularization-Associated Factor

Manipulation or modification of substances involved in the neovascularization-associated factor and the neovascularization system of the present invention is preferably accomplished by genetic manipulation.

In one aspect, composition and method for manipulating a gene by targeting a partial or entire non-coding or coding region of the neovascularization-associated factor may be provided.

In an exemplary embodiment, the composition and method may be used in manipulation or modification of one or more neovascularization regulating genes involved in the formation of a desired neovascularization system. The manipulation or modification may be performed by modification of nucleic acids constituting a gene. As a result of the manipulation, all of knockdown, knockout, and knockin are included.

In an exemplary embodiment, the manipulation may be performed by targeting a promoter region, or a transcription sequence, for example, an intron or exon sequence. A coding sequence, for example, a coding region, specifically, an initial coding region may be targeted for the modification of expression and knockout.

In an exemplary embodiment, the modification of nucleic acids may be substitution, deletion, and/or insertion of one or more nucleotides, for example, 1 to 30 bp, 1 to 27 bp, 1 to 25 bp, 1 to 23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, 1 to 3 bp, or 1 bp nucleotides.

In an exemplary embodiment, for the knockout of one or more neovascularization-associated genes, elimination of expression of one or more of the genes, or one or more knockouts of one or two alleles, the above-mentioned region may be targeted such that one or more neovascularization-associated genes contain a deletion or mutation.

In an exemplary embodiment, the knockdown of a gene may be used to decrease the expression of undesired alleles or transcriptomes.

In an exemplary embodiment, non-coding sequences of a promoter, an enhancer, an intron, a 3′UTR, and/or a polyadenylation signal may be targeted to be used in modifying a neovascularization-associated gene affecting a neovascularization function.

In an exemplary embodiment, the activity of a neovascularization-associated gene may be regulated, for example, activated or inactivated by the modification of nucleic acids of the gene.

In an exemplary embodiment, the modification of nucleic acids of the gene may catalyze cleavage of a single strand or double strands, that is, breaks of nucleic acid strands in a specific region of the target gene by a guide nucleic acid-editor protein complex, resulting in inactivation of the target gene.

In an exemplary embodiment, the nucleic acid strand breaks may be repaired through a mechanism such as homologous recombination or non-homologous end joining (NHEJ).

In this case, when the NHEJ mechanism takes place, a change in DNA sequence is induced at the cleavage site, resulting in inactivation of the gene. The repair by NHEJ may induce substitution, insertion or deletion of a short gene fragment, and may be used in the induction of a corresponding gene knockout.

In another aspect, the present invention provides a composition for manipulating a neovascularization-associated factor.

The composition for manipulation is a composition that is able to artificially manipulate a neovascularization-associated factor, and preferably, a composition for gene manipulation.

The composition may be employed in gene manipulation for one or more neovascularization-associated factors involved in formation of a desired neovascularization-regulating system.

The gene manipulation may be performed in consideration of a gene expression regulating process.

In an exemplary embodiment, it may be performed by selecting a suitable manipulation means for each stage of transcription, RNA processing, RNA transporting, RNA degradation, translation, and protein modification regulating stages.

In an exemplary embodiment, small RNA (sRNA) interferes with mRNA or reduces stability thereof using RNA interference (RNAi) or RNA silencing, and in some cases, breaks up mRNA to interrupt the delivery of protein synthesis information, resulting in regulation of the expression of genetic information.

The gene manipulation may be performed by modification of nucleic acids constituting a neovascularization-associated factor. As manipulation results, all of knockdown, knockout, and knockin are included.

In a certain embodiment, the modification of nucleic acids may be substitution, deletion, and/or insertion of one or more nucleotides, for example, 1 to 30 bp, 1 to 27 bp, 1 to 25 bp, 1 to 23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, 1 to 3 bp, or 1 bp nucleotides.

In a certain embodiment, for knockout of one or more neovascularization-associated factors, elimination of the expression of one or more factors, or one or more knockouts of one or two alleles, the gene may be manipulated such that one or more neovascularization-associated factors contain a deletion or mutation.

In a certain embodiment, knockdown of the neovascularization-associated factor may be used to decrease expression of undesired alleles or transcriptomes.

In a certain embodiment, the modification of nucleic acids may be insertion of one or more nucleic acid fragments or genes. Here, the nucleic acid fragment may be a nucleic acid sequence consisting of one or more nucleotides, and a length of the nucleic acid fragment may be 1 to 40 bp, 1 to 50 bp, 1 to 60 bp, 1 to 70 bp, 1 to 80 bp, 1 to 90 bp, 1 to 100 bp, 1 to 500 bp or 1 to 1000 bp. Here, the inserted gene may be one of the neovascularization-associated factors, or a gene having a different function.

In an exemplary embodiment, the modification of nucleic acids may employ a wild type or variant enzyme which is capable of catalyzing hydrolysis (cleavage) of bonds between nucleic acids in a DNA or RNA molecule, preferably, a DNA molecule. It may also employ a guide nucleic acid-editor protein complex.

For example, the gene may be manipulated using one or more nucleases selected from the group consisting of a meganuclease, a zinc finger nuclease, CRISPR/Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) and a TALE-nuclease, thereby regulating the expression of genetic information.

In a certain embodiment, non-limitedly, the gene manipulation may be mediated by NHEJ or homology-directed repair (HDR) using a guide nucleic acid-editor protein complex, for example, a CRISPR/Cas system.

In this case, when the NHEJ mechanism takes place, a change in DNA sequence may be induced at a cleavage site, thereby inactivating the gene. Repair by NHEJ may induce substitution, insertion or deletion of a short gene fragment, and may be used in the induction of the knockout of a corresponding gene.

In another aspect, the present invention may provide the gene manipulation site.

In an exemplary embodiment, when the gene is modified by NHEJ-mediated modification, the gene manipulation site may be a site in the gene, triggering the decrease or elimination of expression of a neovascularization regulating gene product.

For example, the site may be in an initial coding region,

-   -   a promoter sequence,     -   an enhancer sequence,     -   a specific intron sequence, or     -   a specific exon sequence.

In an exemplary embodiment, the composition for manipulating a neovascularization-associated factor may target a neovascularization-associated factor affecting the regulation of neovascularization, such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, or an ANGPTL4 gene, as a manipulation subject.

Examples of target regions, that is, target sequences for regions in which gene manipulation occurs or which are recognized for gene manipulation are summarized in Table 1, Table 2, Table 3, Table 4 and Table 5.

The target sequence may target one or more genes.

The target sequence may simultaneously target two or more genes. Here, the two or more genes may be homologous genes or heterologous genes.

The gene may contain one or more target sequences.

The gene may be simultaneously targeted at two or more target sequences.

The gene may be changed in the site and number of gene manipulations according to the number of target sequences.

The gene manipulation may be designed in various forms depending on the number and positions of the target sequences.

The gene manipulation may simultaneously occur in two or more target sequences. Here, the two or more target sequences may be present in the homologous gene or heterologous gene.

The gene manipulation may be simultaneously performed with respect to the two or more genes. Here, the two or more genes may be homologous genes or heterologous genes.

Hereinafter, examples of target sequences which are able to be used in embodiments of the present invention are shown in the following tables:

-   -   Table 1 Target sequences of VEGFA gene     -   Table 2 Target sequences of HIF1A gene     -   Table 3 Target sequences of ANGPT2 gene     -   Table 4 Target sequences of EPAS1 gene     -   Table 5 Target sequences of ANGPTL4 gene

TABLE 1 Gene No. Target sequence VEGFA 1 GTAGAGCAGCAAGGCAAGGCTC (SEQ ID NO: 1) VEGFA 2 CTTTCTGTCCTCAGTGGTCCCA (SEQ ID NO: 2) VEGFA 3 GAGACCCGGTGGACATCTTCC (SEQ ID NO: 3) VEGFA 4 TTCCAGGAGTACCCTGATGAGA (SEQ ID NO: 4) VEGFA 5 TTGAAGATGTACTCGATCTCAT (SEQ ID NO: 5) VEGFA 6 AGGGGCACACAGGATGGCTTGA (SEQ ID NO: 6) VEGFA 7 AGCAGCCCCCGCATCGCATCAG (SEQ ID NO: 7) VEGFA 8 GCAGCAGCCCCCGCATCGCATC (SEQ ID NO: 8) VEGFA 9 GTGATGTTGGACTCCTCAGTGG (SEQ ID NO: 9) VEGFA 10 TGGTGATGTTGGACTCCTCAGT (SEQ ID NO: 10) VEGFA 11 CATGGTGATGTTGGACTCCTCA (SEQ ID NO: 11) VEGFA 12 ATGCGGATCAAACCTCACCAAG (SEQ ID NO: 12) VEGFA 13 CACATAGGAGAGATGAGCTTCC (SEQ ID NO: 13)

TABLE 2 Gene No. Target sequence HIF1A 1 ACTCACCAGCATCCAGAAGTTT (SEQ ID NO: 14) HIF1A 2 ATTTGGATATTGAAGATGACAT (SEQ ID NO: 15) HIF1A 3 ATTTACATTTCTGATAATGTGA (SEQ ID NO: 16) HIF1A 4 ATGTGTTTACAGTTTGAACTAA (SEQ ID NO: 17) HIF1A 5 CTGTGTCCAGTTAGTTCAAACT (SEQ ID NO: 18) HIF1A 6 ATGGTCACATGGATGAGTAAAA (SEQ ID NO: 19) HIF1A 7 CATGAGGAAATGAGAGAAATGC (SEQ ID NO: 20) HIF1A 8 CCCAGTGAGAAAAGGGAAAGAA (SEQ ID NO: 21) HIF1A 9 TTGTGAAAAAGGGTAAAGAACA (SEQ ID NO: 22) HIF1A 10 ATAGTTCTTCCTCGGCTAGTTA (SEQ ID NO: 23) HIF1A 11 TCATAGTTCTTCCTCGGCTAGT (SEQ ID NO: 24) HIF1A 12 TGTTCTTCATACACAGGTATTG (SEQ ID NO: 25) HIF1A 13 TACGTGAATGTGGCCTGTGCAG (SEQ ID NO: 26) HIF1A 14 CTGCACAGGCCACATTCACGTA (SEQ ID NO: 27) HIF1A 15 CTGAGGTTGGTTACTGTTGGTA (SEQ ID NO: 28) HIF1A 16 CAGGTCATAGGTGGTTTCTTAT (SEQ ID NO: 29) HIF1A 17 ACCAAGCAGGTCATAGGTGGTT (SEQ ID NO: 30) HIF1A 18 TTAGATAGCAAGACTTTCCTCA (SEQ ID NO: 31)

TABLE 3 Gene No. Target sequence ANGPT2 1 TCAGGTCCAGCATGGGTCCTGC (SEQ ID NO: 32) ANGPT2 2 CGGCGCGTCCCTCTGCACAGCA (SEQ ID NO: 33) ANGPT2 3 GCTGTGCAGAGGGACGCGCCGC (SEQ ID NO: 34) ANGPT2 4 ATCGTATTCGAGCGGCGCGTCC (SEQ ID NO: 35) ANGPT2 5 GATGTTCTCCAGCACTTGCAGC (SEQ ID NO: 36) ANGPT2 6 AGTGCTGGAGAACATCATGGAA (SEQ ID NO: 37) ANGPT2 7 ACAACATGAAGAAAGAAATGGT (SEQ ID NO: 38) ANGPT2 8 AAATGGTAGAGATACAGCAGAA (SEQ ID NO: 39) ANGPT2 9 TTCTATCATCACAGCCGTCTGG (SEQ ID NO: 40) ANGPT2 10 AAGTTCAAGTCTCGTGGTCTGA (SEQ ID NO: 41) ANGPT2 11 ACGAGACTTGAACTTCAGCTCT (SEQ ID NO: 42) ANGPT2 12 AAGAAGGTGCTAGCTATGGAAG (SEQ ID NO: 43) ANGPT2 13 GATGATGTGCTTGTCTTCCATA (SEQ ID NO: 44)

TABLE 4 Gene No. Target sequence EPAS1 1 AACACCTCCGTCTCCTTGCTCC (SEQ ID NO: 45) EPAS1 2 GAAGCTGACCAGCAGATGGACA (SEQ ID NO: 46) EPAS1 3 GCAATGAAACCCTCCAAGGCTT (SEQ ID NO: 47) EPAS1 4 AAAACATCAGCAAGTTCATGGG (SEQ ID NO: 48) EPAS1 5 GCAAGTTCATGGGACTTACACA (SEQ ID NO: 49) EPAS1 6 GGTCGCAGGGATGAGTGAAGTC (SEQ ID NO: 50) EPAS1 7 GCGGGACTTCTTCATGAGGATG (SEQ ID NO: 51) EPAS1 8 GAAGTGCACGGTCACCAACAGA (SEQ ID NO: 52) EPAS1 9 ACAGTACGGCCTCTGTTGGTGA (SEQ ID NO: 53) EPAS1 10 TCCAGGTGGCTGACTTGAGGTT (SEQ ID NO: 54) EPAS1 11 CAGGACAGCAGGGGCTCCTTGT (SEQ ID NO: 55) EPAS1 12 TAGCCCCCATGCTTTGCGAGCA (SEQ ID NO: 56)

TABLE 5 Gene No. Target sequence ANGPTL4 1 GCATCAGGGCTGCCCCGGCCGT (SEQ ID NO: 57) ANGPTL4 2 CACGGGTCCGCCCTGAGCGCTC (SEQ ID NO: 58) ANGPTL4 3 GGACGCAAAGCGCGGCGACTTG (SEQ ID NO: 59) ANGPTL4 4 TCCTGGGACGAGATGAATGTCC (SEQ ID NO: 60) ANGPTL4 5 CTGCAGCTCGGCCAGGGGCTGC (SEQ ID NO: 61) ANGPTL4 6 CCAGGGGCTGCGCGAACACGCG (SEQ ID NO: 62) ANGPTL4 7 CCCTCGGTTCCCTGACAGGCGG (SEQ ID NO: 63) ANGPTL4 8 ACCCTGAGGTCCTTCACAGCCT (SEQ ID NO: 64) ANGPTL4 9 TTCCACAAGGTGGCCCAGCAGC (SEQ ID NO: 65) ANGPTL4 10 CAGCAGCAGCGGCACCTGGAGA (SEQ ID NO: 66) ANGPTL4 11 TCCTAGTTTGGCCTCCTGGACC (SEQ ID NO: 67) ANGPTL4 12 GACCCGGCTCACAATGTCAGCC (SEQ ID NO: 68) ANGPTL4 13 GCTGTTGCGGTCCCCCGTGATG (SEQ ID NO: 69) ANGPTL4 14 GGCGTTGCCATCCCAGTCCCGC (SEQ ID NO: 70) ANGPTL4 15 AACGCCGAGTTGCTGCAGTTCT (SEQ ID NO: 71) ANGPTL4 16 ATAGGCCGTGTCCTCGCCACCC (SEQ ID NO: 72) ANGPTL4 17 GTTCTCCGTGCACCTGGGTGGC (SEQ ID NO: 73) ANGPTL4 18 ACACGGCCTATAGCCTGCAGCT (SEQ ID NO: 74) ANGPTL4 19 CCACCGTCCCACCCAGCGGCCT (SEQ ID NO: 75) ANGPTL4 20 GTGATCCTGGTCCCAAGTGGAG (SEQ ID NO: 76) ANGPTL4 21 GACCCCGGCAGGAGGCTGGTGG (SEQ ID NO: 77) ANGPTL4 22 TGCAGCCATTCCAACCTCAACG (SEQ ID NO: 78) ANGPTL4 23 TGCCGCTGCTGTGGGATGGAGC (SEQ ID NO: 79)

Composition for Manipulation-Gene Scissors System

The neovascularization-regulating system of the present invention may include a guide nucleic acid-editor protein complex as a composition for manipulating a neovascularization-associated factor.

Guide Nucleic Acid-Editor Protein Complex

The term “guide nucleic acid-editor protein complex” refers to a complex formed through the interaction between a guide nucleic acid and an editor protein, and the nucleic acid-protein complex includes a guide nucleic acid and an editor protein.

The term “guide nucleic acid” refers to a nucleic acid capable of recognizing a target nucleic acid, gene, chromosome or protein.

The guide nucleic acid may be present in the form of DNA, RNA or a DNA/RNA hybrid, and may have a nucleic acid sequence of 5 to 150 bases.

The guide nucleic acid may include one or more domains.

The domains may be, but are not limited to, a guide domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, or a tail domain.

The guide nucleic acid may include two or more domains, which may be the same domain repeats, or different domains.

The guide nucleic acid may have one continuous nucleic acid sequence.

For example, the one continuous nucleic acid sequence may be (N)m, where N represents A, T, C or G, or A, U, C or G, and m is an integer of 1 to 150.

The guide nucleic acid may have two or more continuous nucleic acid sequences.

For example, the two or more continuous nucleic acid sequences may be (N)m and (N)o, where N represents A, T, C or G, or A, U, C or G, m and o are an integer of 1 to 150, and m and o may be the same as or different from each other.

The term “editor protein” refers to a peptide, polypeptide or protein which is able to directly bind to or interact with, without direct binding to, a nucleic acid.

The editor protein may be an enzyme.

The editor protein may be a fusion protein.

Here, the “fusion protein” refers to a protein that is produced by fusing an enzyme with an additional domain, peptide, polypeptide or protein.

The term “enzyme” refers to a protein that contains a domain capable of cleaving a nucleic acid, gene, chromosome or protein.

The additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein, which has a function the same as or different from the enzyme.

The fusion protein may include an additional domain, peptide, polypeptide or protein at one or more regions of the amino terminus (N-terminus) of the enzyme or the vicinity thereof; the carboxyl terminus (C-terminus) or the vicinity thereof; the middle part of the enzyme; and a combination thereof.

The fusion protein may include a functional domain, peptide, polypeptide or protein at one or more regions of the N-terminus of the enzyme or the vicinity thereof; the C-terminus or the vicinity thereof; the middle part of the enzyme; and a combination thereof.

The guide nucleic acid-editor protein complex may serve to modify a subject.

The subject may be a target nucleic acid, gene, chromosome or protein.

For example, the guide nucleic acid-editor protein complex may result in final regulation (e.g., inhibition, suppression, reduction, increase or promotion) of the expression of a protein of interest, removal of the protein, or expression of a new protein.

Here, the guide nucleic acid-editor protein complex may act at a DNA, RNA, gene or chromosome level.

The guide nucleic acid-editor protein complex may act in gene transcription and translation stages.

The guide nucleic acid-editor protein complex may act at a protein level.

1. Guide Nucleic Acids

The guide nucleic acid is a nucleic acid that is capable of recognizing a target nucleic acid, gene, chromosome or protein, and forms a guide nucleic acid-protein complex.

Here, the guide nucleic acid is configured to recognize or target a nucleic acid, gene, chromosome or protein targeted by the guide nucleic acid-protein complex.

The guide nucleic acid may be present in the form of DNA, RNA or a DNA/RNA mixture, and have a 5 to 150-nucleic acid sequence.

The guide nucleic acid may be present in a linear or circular shape.

The guide nucleic acid may be one continuous nucleic acid sequence.

For example, the one continuous nucleic acid sequence may be (N)_(m), where N is A, T, C or G, or A, U, C or G, and m is an integer of 1 to 150.

The guide nucleic acid may be two or more continuous nucleic acid sequences.

For example, the two or more continuous nucleic acid sequences may be (N)m and (N)o, where N represents A, T, C or G, or A, U, C or G, m and o are an integer of 1 to 150, and may be the same as or different from each other.

The guide nucleic acid may include one or more domains.

Here, the domains may be, but are not limited to, a guide domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, or a tail domain.

The guide nucleic acid may include two or more domains, which may be the same domain repeats, or different domains.

The domains will be described below.

i) Guide Domain

The term “guide domain” is a domain having a complementary guide sequence which is able to form a complementary bond with a target sequence on a target gene or nucleic acid, and serves to specifically interact with the target gene or nucleic acid.

The guide sequence is a nucleic acid sequence complementary to the target sequence on a target gene or nucleic acid, which has, for example, at least 50% or more, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% complementarity or complete complementarity.

The guide domain may be a sequence of 5 to 50 bases.

In an example, the guide domain may be a sequence of 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50 or 45 to 50 bases.

In another example, the guide domain may be a sequence of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 bases.

The guide domain may have a guide sequence.

The guide sequence may be a complementary base sequence which is able to form a complementary bond with the target sequence on the target gene or nucleic acid.

The guide sequence may be a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

The guide sequence may be a 5 to 50-base sequence.

In an example, the guide domain may be a 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, or 45 to 50-base sequence.

In another example, the guide sequence may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50-base sequence.

In addition, the guide domain may include a guide sequence and an additional base sequence.

The additional base sequence may be utilized to improve or degrade the function of the guide domain.

The additional base sequence may be utilized to improve or degrade the function of the guide sequence.

The additional base sequence may be a 1 to 35-base sequence.

In one example, the additional base sequence may be a 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35 or 30 to 35-base sequence.

In another example, the additional base sequence may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30 or 30 to 35-base sequence.

The additional base sequence may be located at the 5′end of the guide sequence.

The additional base sequence may be located at the 3′end of the guide sequence.

ii) First Complementary Domain

The term “first complementary domain” is a nucleic acid sequence including a nucleic acid sequence complementary to a second complementary domain, and has enough complementarity so as to form a double strand with the second complementary domain.

The first complementary domain may be a 5 to 35-base sequence.

In an example, the first complementary domain may be a 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, or 30 to 35-base sequence.

In another example, the first complementary domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30 or 30 to 35-base sequence.

iii) Linker Domain

The term “linker domain” is a nucleic acid sequence connecting two or more domains, which are two or more identical or different domains. The linker domain may be connected with two or more domains by covalent bonding or non-covalent bonding, or may connect two or more domains by covalent bonding or non-covalent bonding.

The linker domain may be a 1 to 30-base sequence.

In one example, the linker domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30-base sequence.

In another example, the linker domain may be a 1 to 30, 5 to 30, 10 to 30, 15 to 30, 20 to 30, or 25 to 30-base sequence.

iv) Second Complementary Domain

The term “second complementary domain” is a nucleic acid sequence including a nucleic acid sequence complementary to the first complementary domain, and has enough complementarity so as to form a double strand with the first complementary domain.

The second complementary domain may have a base sequence complementary to the first complementary domain, and a base sequence having no complementarity to the first complementary domain, for example, a base sequence not forming a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain.

The second complementary domain may have a 5 to 35-base sequence.

In an example, the second complementary domain may be a 1 to 35, 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, or 30 to 35-base sequence.

In another example, the second complementary domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 30 to 35-base sequence.

v) Proximal Domain

The term “proximal domain” is a nucleic acid sequence located adjacent to the second complementary domain.

The proximal domain may have a complementary base sequence therein, and may be formed in a double strand due to a complementary base sequence.

The proximal domain may be a 1 to 20-base sequence.

In one example, the proximal domain may be a 1 to 20, 5 to 20, 10 to 20 or 15 to 20-base sequence.

In another example, the proximal domain may be a 1 to 5, 5 to 10, 10 to 15 or 15 to 20-base sequence.

vi) Tail Domain

The term “tail domain” is a nucleic acid sequence located at one or more ends of the both ends of the guide nucleic acid.

The tail domain may have a complementary base sequence therein, and may be formed in a double strand due to a complementary base sequence.

The tail domain may be a 1 to 50-base sequence.

In an example, the tail domain may be a 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, or 45 to 50-base sequence.

In another example, the tail domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50-base sequence.

Meanwhile, a part or all of the nucleic acid sequences included in the domains, that is, the guide domain, the first complementary domain, the linker domain, the second complementary domain, the proximal domain and the tail domain may selectively or additionally include a chemical modification.

The chemical modification may be, but is not limited to, methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP).

The guide nucleic acid includes one or more domains.

The guide nucleic acid may include a guide domain.

The guide nucleic acid may include a first complementary domain.

The guide nucleic acid may include a linker domain.

The guide nucleic acid may include a second complementary domain.

The guide nucleic acid may include a proximal domain.

The guide nucleic acid may include a tail domain.

Here, there may be 1, 2, 3, 4, 5, 6 or more domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more guide domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more first complementary domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more linker domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more second complementary domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more proximal domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more tail domains.

Here, in the guide nucleic acid, one type of domain may be duplicated.

The guide nucleic acid may include several domains with or without duplication.

The guide nucleic acid may include the same type of domain. Here, the same type of domain may have the same nucleic acid sequence or different nucleic acid sequences.

The guide nucleic acid may include two types of domains. Here, the two different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

The guide nucleic acid may include three types of domains. Here, the three different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

The guide nucleic acid may include four types of domains. Here, the four different types of domains may have different nucleic acid sequences, or the same nucleic acid sequence.

The guide nucleic acid may include five types of domains. Here, the five different types of domains may have different nucleic acid sequences, or the same nucleic acid sequence.

The guide nucleic acid may include six types of domains. Here, the six different types of domains may have different nucleic acid sequences, or the same nucleic acid sequence.

For example, the guide nucleic acid may consist of [guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[linker domain]-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]. Here, the two guide domains may include guide sequences for different or the same targets, the two first complementary domains and the two second complementary domains may have the same or different nucleic acid sequences. When the guide domains include guide sequences for different targets, the guide nucleic acids may specifically bind to two different targets, and here, the specific bindings may be performed simultaneously or sequentially. In addition, the linker domains may be cleaved by specific enzymes, and the guide nucleic acids may be divided into two or three parts in the presence of specific enzymes.

As a specific example of the guide nucleic acid of the present invention, gRNA will be described below.

gRNA

The term “gRNA” refers to a nucleic acid capable of specifically targeting a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, with respect to a target gene or nucleic acid. In addition, the gRNA is a nucleic acid-specific RNA which may bind to a CRISPR enzyme and guide the CRISPR enzyme to the target gene or nucleic acid.

The gRNA may include multiple domains. Due to each domain, interactions may occur in a three-dimensional structure or active form of a gRNA strand, or between these strands.

The gRNA may be called single-stranded gRNA (single RNA molecule); or double-stranded gRNA (including more than one, generally, two discrete RNA molecules).

In one exemplary embodiment, the single-stranded gRNA may include a guide domain, that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid; a first complementary domain; a linker domain; a second complementary domain, a domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain; a proximal domain; and optionally a tail domain in the 5′ to 3′ direction.

In another embodiment, the double-stranded gRNA may include a first strand which includes a guide domain, that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid and a first complementary domain; and a second strand which includes a second complementary domain, a domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain, a proximal domain; and optionally a tail domain in the 5′ to 3′ direction.

Here, the first strand may be referred to as crRNA, and the second strand may be referred to as tracrRNA. The crRNA may include a guide domain and a first complementary domain, and the tracrRNA may include a second complementary domain, a proximal domain and optionally a tail domain.

In still another embodiment, the single-stranded gRNA may include a guide domain, that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid; a first complementary domain; a second complementary domain, and a domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain in the 5′ to 3′ direction.

i) Guide Domain

The guide domain includes a complementary guide sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid. The guide sequence may be a nucleic acid sequence having complementarity to the target sequence on the target gene or nucleic acid, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity. The guide domain is considered to allow a gRNA-Cas complex, that is, a CRISPR complex to specifically interact with the target gene or nucleic acid.

The guide domain may be a 5 to 50-base sequence.

As an exemplary embodiment, the guide domain may be a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

As an exemplary embodiment, the guide domain may include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

Here, the guide domain may include a guide sequence.

The guide sequence may be a complementary base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid.

The guide sequence may be a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of a neovascularization-associated factor such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene or an ANGPTL4 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

The guide sequence may be a 5 to 50-base sequence.

In an exemplary embodiment, the guide sequence may be a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the VEGFA gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the HIF1A gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the ANGPT2 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the EPAS1 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the ANGPTL4 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

Here, target sequences of the target genes, that is, the neovascularization-associated factors such as the VEGFA gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and the ANGPTL4 gene for the guide sequence are listed above in Table 1, Table 2, Table 3, Table 4 and Table 5, respectively, but the present invention is not limited thereto.

Here, the guide domain may include a guide sequence and an additional base sequence.

The additional base sequence may be a 1 to 35-base sequence.

In one exemplary embodiment, the additional base sequence may be a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-base sequence.

For example, the additional base sequence may be a single base sequence, guanine (G), or a sequence of two bases, GG.

The additional base sequence may be located at the 5′ end of the guide sequence.

The additional base sequence may be located at the 3′ end of the guide sequence.

Selectively, a part or all of the base sequence of the guide domain may include a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′ phosphorothioate (MS) or 2′-O-methyl 3′ thioPACE (MSP), but the present invention is not limited thereto.

ii) First Complementary Domain

The first complementary domain includes a nucleic acid sequence complementary to a second complementary domain, and has enough complementarity such that it is able to form a double strand with the second complementary domain.

Here, the first complementary domain may be a 5 to 35-base sequence. The first complementary domain may include a 5 to 35-base sequence.

In one exemplary embodiment, the first complementary domain may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-base sequence.

In another embodiment, the first complementary domain may include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-base sequence.

The first complementary domain may have homology with a natural first complementary domain, or may be derived from a natural first complementary domain. In addition, the first complementary domain may have a difference in the base sequence of a first complementary domain depending on the species existing in nature, may be derived from a first complementary domain contained in the species existing in nature, or may have partial or complete homology with the first complementary domain contained in the species existing in nature.

In one exemplary embodiment, the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus or Neisseria meningitides, or a first complementary domain derived therefrom.

For example, when the first complementary domain is the first complementary domain of Streptococcus pyogenes or a first complementary domain derived therefrom, the first complementary domain may be 5′-GUUUUAGAGCUA-3′ or a base sequence having partial, that is, at least 50% or more, or complete homology with 5′-GUUUUAGAGCUA-3′. Here, the first complementary domain may further include (X)n, resulting in 5′-GUUUUAGAGCUA(X)n-3′. The X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 5 to 15. Here, the (X)n may be n repeats of the same base, or a mixture of n bases of A, T, U and G.

In another embodiment, when the first complementary domain is the first complementary domain of Campylobacter jejuni or a first complementary domain derived therefrom, the first complementary domain may be 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′, or a base sequence having partial, that is, at least 50% or more, or complete homology with 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′. Here, the first complementary domain may further include (X)n, resulting in 5′-GUUUUAGUCCCUUUUUAAAUUUCUU(X)n-3′. The X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 5 to 15. Here, the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.

In another embodiment, the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium eligens, or a first complementary domain derived therefrom.

For example, when the first complementary domain is the first complementary domain of Parcubacteria bacterium or a first complementary domain derived therefrom, the first complementary domain may be 5′-UUUGUAGAU-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-UUUGUAGAU-3′. Here, the first complementary domain may further include (X)n, resulting in 5′-(X)nUUUGUAGAU-3′. The X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 5. Here, the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.

Selectively, a part or all of the base sequence of the first complementary domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′ phosphorothioate (MS) or 2′-O-methyl 3′ thioPACE (MSP), but the present invention is not limited thereto.

iii) Linker Domain

The linker domain is a nucleic acid sequence connecting two or more domains, and connects two or more identical or different domains. The linker domain may be connected with two or more domains, or may connect two or more domains by covalent or non-covalent bonding.

The linker domain may be a nucleic acid sequence connecting a first complementary domain with a second complementary domain to produce single-stranded gRNA.

The linker domain may be connected with the first complementary domain and the second complementary domain by covalent or non-covalent bonding.

The linker domain may connect the first complementary domain with the second complementary domain by covalent or non-covalent bonding

The linker domain may be a 1 to 30-base sequence. The linker domain may include a 1 to 30-base sequence.

In an exemplary embodiment, the linker domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25 or 25 to 30-base sequence.

In an exemplary embodiment, the linker domain may include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30-base sequence.

The linker domain is suitable to be used in a single-stranded gRNA molecule, and may be used to produce single-stranded gRNA by being connected with a first strand and a second strand of double-stranded gRNA or connecting the first strand with the second strand by covalent or non-covalent bonding. The linker domain may be used to produce single-stranded gRNA by being connected with crRNA and tracrRNA of double-stranded gRNA or connecting the crRNA with the tracrRNA by covalent or non-covalent bonding.

The linker domain may have homology with a natural sequence, for example, a partial sequence of tracrRNA, or may be derived therefrom.

Selectively, a part or all of the base sequence of the linker domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′ phosphorothioate (MS) or 2′-O-methyl 3′ thioPACE (MSP), but the present invention is not limited thereto.

iv) Second Complementary Domain

The second complementary domain includes a nucleic acid sequence complementary to the first complementary domain, and has enough complementarity so as to form a double strand with the first complementary domain. The second complementary domain may include a base sequence complementary to the first complementary domain, and a base sequence having no complementarity with the first complementary domain, for example, a base sequence not forming a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain.

Here, the second complementary domain may be a 5 to 35-base sequence. The first complementary domain may include a 5 to 35-base sequence.

In an exemplary embodiment, the second complementary domain may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In an exemplary embodiment, the second complementary domain may include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In addition, the second complementary domain may have homology with a natural second complementary domain, or may be derived from the natural second complementary domain. In addition, the second complementary domain may have a difference in base sequence of a second complementary domain according to a species existing in nature, and may be derived from a second complementary domain contained in the species existing in nature, or may have partial or complete homology with the second complementary domain contained in the species existing in nature.

In an exemplary embodiment, the second complementary domain may have partial, that is, at least 50% or more, or complete homology with a second complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus or Neisseria meningitides, or a second complementary domain derived therefrom.

For example, when the second complementary domain is a second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, the second complementary domain may be 5′-UAGCAAGUUAAAAU-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-UAGCAAGUUAAAAU-3′ (a base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further include (X)n and/or (X)m, resulting in 5′-(X)n UAGCAAGUUAAAAU(X)m-3′. The X may be selected from the group consisting of bases A, T, U and G, and each of the n and m may represent the number of bases, in which the n may be an integer of 1 to 15, and the m may be an integer of 1 to 6. Here, the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G. In addition, (X)m may represent m repeats of the same base, or a mixture of m bases of A, T, U and G.

In another example, when the second complementary domain is the second complementary domain of Campylobacter jejuni or a second complementary domain derived therefrom, the second complementary domain may be 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′ (a base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further include (X)n and/or (X)m, resulting in 5′-(X)nAAGAAAUUUAAAAAGGGACUAAAAU(X)m-3′. The X may be selected from the group consisting of bases A, T, U and G, and each of the n and m may represent the number of bases, in which the n may be an integer of 1 to 15, and the m may be an integer of 1 to 6. Here, (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G. In addition, (X)m may represent m repeats of the same base, or a mixture of m bases of A, T, U and G.

In another embodiment, the secondcomplementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium eligens, or a secondcomplementary domain derived therefrom.

For example, when the second complementary domain is a second complementary domain of Parcubacteria bacterium or a second complementary domain derived therefrom, the second complementary domain may be 5′-AAAUUUCUACU-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-AAAUUUCUACU-3′ (a base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further include (X)n and/or (X)m, resulting in 5′-(X)nAAAUUUCUACU(X)m-3′. The X may be selected from the group consisting of bases A, T, U and G, and each of the n and m may represent the number of bases, in which the n may be an integer of 1 to 10, and the m may be an integer of 1 to 6. Here, the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G. In addition, the (X)m may represent m repeats of the same base, or a mixture of m bases of A, T, U and G.

Selectively, a part or all of the base sequence of the second complementary domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.

v) Proximal Domain

The proximal domain is a sequence of 1 to 20 bases located adjacent to the second complementary domain, and a domain located at the 3′end direction of the second complementary domain. Here, the proximal domain may be used to form a double strand between complementary base sequences therein.

In one exemplary embodiment, the proximal domain may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-base sequence.

In another embodiment, the proximal domain may include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-base sequence.

In addition, the proximal domain may have homology with a natural proximal domain, or may be derived from the natural proximal domain. In addition, the proximal domain may have a difference in base sequence according to a species existing in nature, may be derived from a proximal domain contained in the species existing in nature, or may have partial or complete homology with the proximal domain contained in the species existing in nature.

In an exemplary embodiment, the proximal domain may have partial, that is, at least 50% or more, or complete homology with a proximal domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus or Neisseria meningitides, or a proximal domain derived therefrom.

For example, when the proximal domain is a proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, the proximal domain may be 5′-AAGGCUAGUCCG-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-AAGGCUAGUCCG-3′. Here, the proximal domain may further include (X)n, resulting in 5′-AAGGCUAGUCCG(X)n-3′. The X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 15. Here, the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.

In yet another example, when the proximal domain is a proximal domain of Campylobacter jejuni or a proximal domain derived therefrom, the proximal domain may be 5′-AAAGAGUUUGC-3′, or a base sequence having at least 50% or more homology with 5′-AAAGAGUUUGC-3′. Here, the proximal domain may further include (X)n, resulting in 5′-AAAGAGUUUGC(X)n-3′. The X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 40. Here, the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.

Selectively, a part or all of the base sequence of the proximal domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.

vi) Tail Domain

The tail domain is a domain which is able to be selectively added to the 3′ end of single-stranded gRNA or double-stranded gRNA. The tail domain may be a 1 to 50-base sequence, or include a 1 to 50-base sequence. Here, the tail domain may be used to form a double strand between complementary base sequences therein.

In an exemplary embodiment, the tail domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50-base sequence.

In an exemplary embodiment, the tail domain may include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50-base sequence.

In addition, the tail domain may have homology with a natural tail domain, or may be derived from the natural tail domain. In addition, the tail domain may have a difference in base sequence according to a species existing in nature, may be derived from a tail domain contained in a species existing in nature, or may have partial or complete homology with a tail domain contained in a species existing in nature.

In one exemplary embodiment, the tail domain may have partial, that is, at least 50% or more, or complete homology with a tail domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus or Neisseria meningitides or a tail domain derived therefrom.

For example, when the tail domain is a tail domain of Streptococcus pyogenes or a tail domain derived therefrom, the tail domain may be 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′. Here, the tail domain may further include (X)_(n), resulting in 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X)_(n)-3′. The X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 15. Here, the (X), may represent n repeats of the same base, or a mixture of n bases such as A, T, U and G.

In another example, when the tail domain is a tail domain of Campylobacter jejuni or a tail domain derived therefrom, the tail domain may be 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′. Here, the tail domain may further include (X)n, resulting in 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU(X)n-3′. The X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 15. Here, the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.

In another embodiment, the tail domain may include a 1 to 10-base sequence at the 3′ end involved in an in vitro or in vivo transcription method.

For example, when a T7 promoter is used in in vitro transcription of gRNA, the tail domain may be an arbitrary base sequence present at the 3′ end of a DNA template. In addition, when a U6 promoter is used in in vivo transcription, the tail domain may be UUUUUU, when an H1 promoter is used in transcription, the tail domain may be UUUU, and when a pol-III promoter is used, the tail domain may include several uracil bases or alternative bases.

Selectively, a part or all of the base sequence of the tail domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.

The gRNA may include a plurality of domains as described above, and therefore, the length of the nucleic acid sequence may be regulated according to a domain contained in the gRNA, and interactions may occur in strands in a three-dimensional structure or active form of gRNA or between theses strands due to each domain.

The gRNA may be referred to as single-stranded gRNA (single RNA molecule); or double-stranded gRNA (including more than one, generally two discrete RNA molecules).

Double-Stranded gRNA

The double-stranded gRNA consists of a first strand and a second strand.

Here, the first strand may consist of 5′-[guide domain]-[first complementary domain]-3′, and the second strand may consist of 5′-[second complementary domain]-[proximal domain]-3′ or 5′-[second complementary domain]-[proximal domain]-[tail domain]-3′.

Here, the first strand may be referred to as crRNA, and the second strand may be referred to as tracrRNA.

First Strand

Guide Domain

In the first strand, the guide domain includes a complementary guide sequence which is able to form a complementary bond with a target sequence on a target gene or nucleic acid. The guide sequence is a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity. The guide domain is considered to allow a gRNA-Cas complex, that is, a CRISPR complex to specifically interact with the target gene or nucleic acid.

Here, the guide domain may be a 5 to 50-base sequence, or includes a 5 to 50-base sequence. For example, the guide domain may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In addition, the guide domain may include a guide sequence.

Here, the guide sequence may be a complementary base sequence which is able to form a complementary bond with a target sequence on a target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

In an exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of a neovascularization-associated factor such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, or an ANGPTL4 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

Here, the guide sequence may be a 5 to 50-base sequence or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence is a nucleic acid sequence complementary to a target sequence of the VEGFA gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the HIF1A gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the ANGPT2 gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence is a nucleic acid sequence complementary to a target sequence of the EPAS1 gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence is a nucleic acid sequence complementary to a target sequence of the ANGPTL4 gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

Here, for the guide sequence, target genes, that is, target sequences of neovascularization-associated factors such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene are listed above in Table 1, Table 2, Table 3, Table 4 and Table 5, but the present invention is not limited thereto.

Selectively, the guide domain may include a guide sequence and an additional base sequence.

Here, the additional base sequence may be a 1 to 35-base sequence. For example, the additional base sequence may be a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-base sequence.

In one exemplary embodiment, the additional base sequence may include one base, guanine (G), or two bases, GG.

Here, the additional base sequence may be located at the 5′ end of the guide domain, or at the 5′ end of the guide sequence.

The additional base sequence may be located at the 3′ end of the guide domain, or at the 3′ end of the guide sequence.

First Complementary Domain

The first complementary domain includes a nucleic acid sequence complementary to a second complementary domain of the second strand, and is a domain having enough complementarity so as to form a double strand with the second complementary domain.

Here, the first complementary domain may be or include a 5 to 35-base sequence. For example, the first complementary domain may be or include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

The first complementary domain may have homology with a natural first complementary domain, or may be derived from a natural first complementary domain. In addition, the first complementary domain may have a difference in base sequence according to a species existing in nature, may be derived from the first complementary domain contained in the species existing in nature, or may have partial or complete homology with the first complementary domain contained in the species existing in nature.

In one exemplary embodiment, the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus or Neisseria meningitides, or a first complementary domain derived therefrom.

Selectively, the first complementary domain may include an additional base sequence which does not undergo complementary bonding with the second complementary domain of the second strand.

Here, the additional base sequence may be a sequence of 1 to 15 bases. For example, the additional base sequence may be a sequence of 1 to 5, 5 to 10, or 10 to 15 bases.

Selectively, a part or all of the base sequence of the guide domain and/or first complementary domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′ phosphorothioate (MS) or 2′-O-methyl 3′ thioPACE (MSP), but the present invention is not limited thereto.

Therefore, the first strand may consist of 5′-[guide domain]-[first complementary domain]-3′ as described above.

In addition, the first strand may optionally include an additional base sequence.

In one example, the first strand may be 5′-(N_(target))-(Q)_(m)-3′; or 5′-(X)_(a)-(N_(target))-(X)_(b)-(Q)_(m)-(X)_(c)-3′.

Here, the Ntarget is a base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, and a base sequence region which may be changed according to a target sequence on a target gene or nucleic acid.

In one exemplary embodiment, Ntarget may be a base sequence capable of forming a complementary bond with a target gene, that is, a target sequence of a neovascularization-associated factor such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene or an ANGPTL4 gene.

Here, the (Q)m is a base sequence including the first complementary domain, which is able to form a complementary bond with the second complementary domain of the second strand. The (Q)m may be a sequence having partial or complete homology with the first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin. The Q may be each independently selected from the group consisting of A, U, C and G, and the m may be the number of bases, which is an integer of 5 to 35.

For example, when the first complementary domain has partial or complete homology with a first complementary domain of Streptococcus pyogenes or a Streptococcus pyogenes-derived first complementary domain, the (Q)m may be 5′-GUUUUAGAGCUA-3′, or a base sequence having at least 50% or more homology with 5′-GUUUUAGAGCUA-3′.

In another example, when the first complementary domain has partial or complete homology with a first complementary domain of Campylobacter jejuni or a Campylobacter jejuni-derived first complementary domain, the (Q)m may be 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′, or a base sequence having at least 50% or more homology with 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′.

In still another example, when the first complementary domain has partial or complete homology with a first complementary domain of Streptococcus thermophilus or a Streptococcus thermophilus-derived first complementary domain, the (Q)m may be 5′-GUUUUAGAGCUGUGUUGUUUCG-3′, or a base sequence having at least 50% or more homology with 5′-GUUUUAGAGCUGUGUUGUUUCG-3′.

In addition, each of the (X)a, (X)b and (X)c is selectively an additional base sequence, where the X may be each independently selected from the group consisting of A, U, C and G, and each of the a, b and c may be the number of bases, which is 0 or an integer of 1 to 20.

Second Strand

The second strand may consist of a second complementary domain and a proximal domain, and selectively include a tail domain.

Second Complementary Domain

In the second strand, the second complementary domain includes a nucleic acid sequence complementary to the first complementary domain of the first strand, and has enough complementarity so as to form a double strand with the first complementary domain. The second complementary domain may include a base sequence complementary to the first complementary domain and a base sequence not complementary to the first complementary domain, for example, a base sequence not forming a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain.

Here, the second complementary domain may be a 5 to 35-base sequence, or include a 5 to 35-base sequence. For example, the second complementary domain may be or include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence, but the present invention is not limited thereto.

The second complementary domain may have homology with a natural second complementary domain, or may be derived from a natural second complementary domain. In addition, the second complementary domain may have a difference in base sequence thereof according to a species existing in nature, may be derived from a second complementary domain contained in the species existing in nature, or may have partial or complete homology with the second complementary domain contained in the species existing in nature.

In one exemplary embodiment, the second complementary domain may have partial, that is, at least 50% or more, or complete homology with a second complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus or Neisseria meningitides, or a second complementary domain derived therefrom.

Selectively, the second complementary domain may further include an additional base sequence which does not undergo complementary bonding with the first complementary domain of the first strand.

Here, the additional base sequence may be a 1 to 25-base sequence. For example, the additional base sequence may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20 or 20 to 25-base sequence.

Proximal Domain

In the second strand, the proximal domain is a sequence of 1 to 20 bases, and a domain located at the 3′ end direction of the second complementary domain. For example, the proximal domain may be or include a sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 bases.

Here, the proximal domain may have a double strand bond between complementary base sequences therein.

In addition, the proximal domain may have homology with a natural proximal domain, or may be derived from a natural proximal domain. In addition, the proximal domain may have a difference in base sequence according to a species existing in nature, may be derived from a proximal domain of a species existing in nature, or may have partial or complete homology with the proximal domain of a species existing in nature.

In one exemplary embodiment, the proximal domain may have partial, that is, at least 50% or more, or complete homology with a proximal domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus or Neisseria meningitides, or a proximal domain derived therefrom.

Tail Domain

Selectively, in the second strand, the tail domain may be a domain selectively added to the 3′ end of the second strand, and the tail domain may be or include a 1 to 50-base sequence. For example, the tail domain may be or include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45 or 45 to 50-base sequence.

Here, the tail domain may have a double strand bond between complementary base sequences therein.

In addition, the tail domain may have homology with a natural tail domain, or may be derived from a natural tail domain. In addition, the tail domain may have a difference in base sequence according to a species existing in nature, may be derived from a tail domain contained in the species existing in nature, or may have partial or complete homology with the tail domain contained in the species existing in nature.

In one exemplary embodiment, the tail domain may have partial, that is, at least 50% or more, or complete homology with a tail domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus or Neisseria meningitides, or a tail domain derived therefrom.

In another embodiment, the tail domain may include a sequence of 1 to 10 bases at the 3′ end involved in an in vitro or in vivo transcription method.

For example, when a T7 promoter is used in in vitro transcription of gRNA, the tail domain may be an arbitrary base sequence present at the 3′ end of a DNA template. In addition, when a U6 promoter is used in in vivo transcription, the tail domain may be UUUUUU, when an H1 promoter is used in transcription, the tail domain may be UUUU, and when a pol-III promoter is used, the tail domain may include several uracil bases or alternative bases.

Selectively, a part or all of each of the base sequence of the second complementary domain, the proximal domain and/or the tail domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.

Therefore, the second strand may consist of 5′-[second complementary domain]-[proximal domain]-3′ or 5′-[second complementary domain]-[proximal domain]-[tail domain]-3′ as described above.

In addition, the second strand may selectively include an additional base sequence.

In one exemplary embodiment, the second strand may be 5′-(Z)h-(P)k-3′; or 5′-(X)d-(Z)h-(X)e-(P)k-(X)f-3′.

In another embodiment, the second strand may be 5′-(Z)h-(P)k-(F)i-3′; or 5′-(X)d-(Z)h-(X)e-(P)k-(X)f-(F)i-3′.

Here, the (Z)h is a base sequence including a second complementary domain, which is able to form a complementary bond with the first complementary domain of the first strand. The (Z)h may be a sequence having partial or complete homology with the second complementary domain of a species existing in nature, and the base sequence of the second complementary domain may be modified according to the species of origin. The Z may be each independently selected from the group consisting of A, U, C and G, and the h may be the number of bases, which is an integer of 5 to 50.

For example, when the second complementary domain has partial or complete homology with a second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, the (Z)h may be 5′-UAGCAAGUUAAAAU-3′, or a base sequence having at least 50% or more homology with 5′-UAGCAAGUUAAAAU-3′.

In another example, when the second complementary domain has partial or complete homology with a second complementary domain of Campylobacter jejuni or a second complementary domain derived therefrom, the (Z)h may be 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′, or a base sequence having at least 50% or more homology with 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′.

In still another example, when the second complementary domain has partial or complete homology with a second complementary domain of Streptococcus thermophilus or a second complementary domain derived therefrom, the (Z)h may be 5′-CGAAACAACACAGCGAGUUAAAAU-3′, or a base sequence having at least 50% or more homology with 5′-CGAAACAACACAGCGAGUUAAAAU-3′.

The (P)k is a base sequence including a proximal domain, which may have partial or complete homology with a proximal domain of a species existing in nature, and the base sequence of the proximal domain may be modified according to the species of origin. The P may be each independently selected from the group consisting of A, U, C and G, and the k may be the number of bases, which is an integer of 1 to 20.

For example, when the proximal domain has partial or complete homology with a proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, the (P)_(k) may be 5′-AAGGCUAGUCCG-3′, or a base sequence having at least 50% or more homology with 5′-AAGGCUAGUCCG-3′.

In another example, when the proximal domain has partial or complete homology with a proximal domain of Campylobacter jejuni or a proximal domain derived therefrom, the (P)_(k) may be 5′-AAAGAGUUUGC-3′, or a base sequence having at least 50% or more homology with 5′-AAAGAGUUUGC-3′.

In still another example, when the proximal domain has partial or complete homology with a proximal domain of Streptococcus thermophilus or a proximal domain derived therefrom, the (P)k may be 5′-AAGGCUUAGUCCG-3′, or a base sequence having at least 50% or more homology with 5′-AAGGCUUAGUCCG-3′.

The (F)i may be a base sequence including a tail domain, and having partial or complete homology with a tail domain of a species existing in nature, and the base sequence of the tail domain may be modified according to the species of origin. The F may be each independently selected from the group consisting of A, U, C and G, and the i may be the number of bases, which is an integer of 1 to 50.

For example, when the tail domain has partial or complete homology with a tail domain of Streptococcus pyogenes or a tail domain derived therefrom, the (F)i may be 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′, or a base sequence having at least 50% or more homology with 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′.

In another example, when the tail domain has partial or complete homology with a tail domain of Campylobacter jejuni or a tail domain derived therefrom, the (F)i may be 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′, or a base sequence having at least 50% or more homology with 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′.

In still another example, when the tail domain has partial or complete homology with a tail domain of Streptococcus thermophilus or a tail domain derived therefrom, the (F)i may be 5′-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3′, or a base sequence having at least 50% or more homology with 5′-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3′.

In addition, the (F)i may include a sequence of 1 to 10 bases at the 3′ end involved in an in vitro or in vivo transcription method.

For example, when a T7 promoter is used in in vitro transcription of gRNA, the tail domain may be an arbitrary base sequence present at the 3′ end of a DNA template. In addition, when a U6 promoter is used in in vivo transcription, the tail domain may be UUUUUU, when an H1 promoter is used in transcription, the tail domain may be UUUU, and when a pol-III promoter is used, the tail domain may include several uracil bases or alternative bases.

In addition, the (X)d, (X)e and (X)f may be base sequences selectively added, where the X may be each independently selected from the group consisting of A, U, C and G, and each of the d, e and f may be the number of bases, which is 0 or an integer of 1 to 20.

Single-Stranded gRNA

Single-stranded gRNA may be classified into two types.

i) Single-Stranded gRNA

First, there is single-stranded gRNA in which a first strand or a second strand of the double-stranded gRNA is linked by a linker domain, and here, the single-stranded gRNA consists of 5′-[first strand]-[linker domain]-[second strand]-3′.

Specifically, the single-stranded gRNA may consist of 5′-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-3′ or 5′-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-[tail domain]-3′.

Each domain except the linker domain is the same as the description of each domain of the first and second strands of the double-stranded gRNA.

Linker Domain

In the single-stranded gRNA, the linker domain is a domain connecting a first strand and a second strand, and specifically, is a nucleic acid sequence which connects a first complementary domain with a second complementary domain to produce single-stranded gRNA. Here, the linker domain may be connected with the first complementary domain and the second complementary domain or connect the first complementary domain with the second complementary domain by covalent or non-covalent bonding.

The linker domain may be or include a 1 to 30-base sequence. For example, the linker domain may be or include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25 or 25 to 30-base sequence.

The linker domain is suitable to be used in a single-stranded gRNA molecule, and may be connected with the first strand and the second strand of the double-stranded gRNA, or connect the first strand with the second strand by covalent or non-covalent bonding to be used in production of the single-stranded gRNA. The linker domain may be connected with crRNA and tracrRNA of the double-stranded gRNA, or connect crRNA with tracrRNA by covalent or non-covalent bonding to be used in production of the single-stranded gRNA.

The linker domain may have homology with a natural sequence, for example, a partial sequence of tracrRNA, or may be derived therefrom.

Selectively, a part or all of the base sequence of the linker domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.

Therefore, the single-stranded gRNA may consist of 5′-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-3′ or 5′-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-[tail domain]-3′ as described above.

In addition, the single-stranded gRNA may selectively include an additional base sequence.

In one exemplary embodiment, the single-stranded gRNA may be

5′-(N_(target))-(Q)_(m)-(L)_(j)-(Z)_(h)-(P)_(k)-3′; or

5′-(N_(target))-(Q)_(m)-(L)_(j)-(Z)_(h)-(P)_(k)-(F)_(i)-3′.

In another embodiment, the single-stranded gRNA may be

5′-(X)_(a)-(N_(target))-(X)_(b)-(Q)_(m)-(X)_(c)-(L)_(j)-(X)_(d)-(Z)_(h)-(X)_(e)-(P)_(k)-(X)_(f)-3′; or

5′-(X)_(a)-(N_(target))-(X)_(b)-(Q)_(m)-(X)_(c)-(L)_(j)-(X)_(d)-(Z)_(h)-(X)_(e)-(P)_(k)-(X)_(f)-(F)_(i)-3′.

Here, the N_(target) is a base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, and a base sequence region capable of being changed according to a target sequence on a target gene or nucleic acid.

In one exemplary embodiment, Ntarget is a base sequence capable of forming a complementary bond with a target gene, that is, a target sequence of a neovascularization-associated factor such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, or an ANGPTL4 gene.

The (Q)m includes a base sequence including the first complementary domain, which is able to form a complementary bond with a second complementary domain. The (Q)m may be a sequence having partial or complete homology with a first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin. The Q may be each independently selected from the group consisting of A, U, C and G, and the m may be the number of bases, which is an integer of 5 to 35.

For example, when the first complementary domain has partial or complete homology with a first complementary domain of Streptococcus pyogenes or a first complementary domain derived therefrom, the (Q)m may be 5′-GUUUUAGAGCUA-3′, or a base sequence having at least 50% or more homology with 5′-GUUUUAGAGCUA-3′.

In another example, when the first complementary domain has partial or complete homology with a first complementary domain of Campylobacter jejuni or a first complementary domain derived therefrom, the (Q)m may be 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′, or a base sequence having at least 50% or more homology with 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′.

In still another example, when the first complementary domain has partial or complete homology with a first complementary domain of Streptococcus thermophilus or a first complementary domain derived therefrom, the (Q)m may be 5′-GUUUUAGAGCUGUGUUGUUUCG-3′, or a base sequence having at least 50% or more homology with 5′-GUUUUAGAGCUGUGUUGUUUCG-3′.

In addition, the (L)j is a base sequence including the linker domain, and connecting the first complementary domain with the second complementary domain, thereby producing single-stranded gRNA. Here, the L may be each independently selected from the group consisting of A, U, C and G, and the j may be the number of bases, which is an integer of 1 to 30.

The (Z)h is a base sequence including the second complementary domain, which is able to have a complementary bond with the first complementary domain. The (Z)h may be a sequence having partial or complete homology with the second complementary domain of a species existing in nature, and the base sequence of the second complementary domain may be changed according to the species of origin. The Z may be each independently selected from the group consisting of A, U, C and G, and the h is the number of bases, which may be an integer of 5 to 50.

For example, when the second complementary domain has partial or complete homology with a second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, the (Z)h may be 5′-UAGCAAGUUAAAAU-3′, or a base sequence having at least 50% or more homology with 5′-UAGCAAGUUAAAAU-3′.

In another example, when the second complementary domain has partial or complete homology with a second complementary domain of Campylobacter jejuni or a second complementary domain derived therefrom, the (Z)h may be 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′, or a base sequence having at least 50% or more homology with 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′.

In still another example, when the second complementary domain has partial or complete homology with a second complementary domain of Streptococcus thermophilus or a second complementary domain derived therefrom, the (Z)h may be 5′-CGAAACAACACAGCGAGUUAAAAU-3′, or a base sequence having at least 50% or more homology with 5′-CGAAACAACACAGCGAGUUAAAAU-3′.

The (P)k is a base sequence including a proximal domain, which may have partial or complete homology with a proximal domain of a species existing in nature, and the base sequence of the proximal domain may be modified according to the species of origin. The P may be each independently selected from the group consisting of A, U, C and G, and the k may be the number of bases, which is an integer of 1 to 20.

For example, when the proximal domain has partial or complete homology with a proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, the (P)k may be 5′-AAGGCUAGUCCG-3′, or a base sequence having at least 50% or more homology with 5′-AAGGCUAGUCCG-3′.

In another example, when the proximal domain has partial or complete homology with a proximal domain of Campylobacter jejuni or a proximal domain derived therefrom, the (P)k may be 5′-AAAGAGUUUGC-3′, or a base sequence having at least 50% or more homology with 5′-AAAGAGUUUGC-3′.

In still another example, when the proximal domain has partial or complete homology with a proximal domain of Streptococcus thermophilus or a proximal domain derived therefrom, the (P)k may be 5′-AAGGCUUAGUCCG-3′, or a base sequence having at least 50% or more homology with 5′-AAGGCUUAGUCCG-3′.

The (F)_(i) may be a base sequence including a tail domain, and having partial or complete homology with a tail domain of a species existing in nature, and the base sequence of the tail domain may be modified according to the species of origin. The F may be each independently selected from the group consisting of A, U, C and G, and the i may be the number of bases, which is an integer of 1 to 50.

For example, when the tail domain has partial or complete homology with a tail domain of Streptococcus pyogenes or a tail domain derived therefrom, the (F)i may be 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′, or a base sequence having at least 50% or more homology with 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′

In another example, when the tail domain has partial or complete homology with a tail domain of Campylobacter jejuni or a tail domain derived therefrom, the (F), may be 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′, or a base sequence having at least 50% or more homology with 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′.

In still another example, when the tail domain has partial or complete homology with a tail domain of Streptococcus thermophilus or a tail domain derived therefrom, the (F), may be 5′-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3′, or a base sequence having at least 50% or more homology with 5′-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3′.

In addition, the (F)i may include a sequence of 1 to 10 bases at the 3′ end involved in an in vitro or in vivo transcription method.

For example, when a T7 promoter is used in in vitro transcription of gRNA, the tail domain may be an arbitrary base sequence present at the 3′ end of a DNA template. In addition, when a U6 promoter is used in in vivo transcription, the tail domain may be UUUUUU, when an H1 promoter is used in transcription, the tail domain may be UUUU, and when a pol-III promoter is used, the tail domain may include several uracil bases or alternative bases.

In addition, the (X)a, (X)b, (X)c, (X)d, (X)e and (X)f may be base sequences selectively added, where the X may be each independently selected from the group consisting of A, U, C and G, and each of the a, b, c, d, e and f may be the number of bases, which is 0 or an integer of 1 to 20.

Single-Stranded gRNA

Second, the single-stranded gRNA may be single-stranded gRNA consisting of a guide domain, a first complementary domain and a second complementary domain, and here, the single-stranded gRNA may consist of: 5′-[second complementary domain]-[first complementary domain]-[guide domain]-3′; or 5′-[second complementary domain]-[linker domain]-[first complementary domain]-[guide domain]-3′.

Guide Domain

In the single-stranded gRNA, the guide domain includes a complementary guide sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid. The guide sequence may be a nucleic acid sequence having complementarity to the target sequence on the target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity. The guide domain is considered to allow a gRNA-Cas complex, that is, a CRISPR complex to specifically interact with the target gene or nucleic acid.

Here, the guide domain may be or include a 5 to 50-base sequence. For example, the guide domain may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In addition, the guide domain may include a guide sequence.

Here, the guide sequence may be a complementary base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of a neovascularization-associated factor such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene or an ANGPTL4 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

Here, the guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the VEGFA gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the HIF1A gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the ANGPT2 gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the EPAS1 gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

In one exemplary embodiment, the guide sequence may be a nucleic acid sequence complementary to a target sequence of the ANGPTL4 gene. The guide sequence may be or include a 5 to 50-base sequence. For example, the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

Here, target sequences of the target genes, that is, the neovascularization-associated factors such as the VEGFA gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and the ANGPTL4 gene for the guide sequence are listed above in Table 1, Table 2, Table 3, Table 4 and Table 5, respectively, but the present invention is not limited thereto.

Selectively, the guide domain may include a guide sequence and an additional base sequence.

Here, the additional base sequence may be a 1 to 35-base sequence. For example, the additional base sequence may be a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-base sequence.

In one exemplary embodiment, the additional base sequence may be a single base sequence, guanine (G), or a sequence of two bases, GG.

Here, the additional base sequence may be located at the 5′ end of the guide domain, or at the 5′ end of the guide sequence.

The additional base sequence may be located at the 3′ end of the guide domain, or at the 3′ end of the guide sequence.

First Complementary Domain

The first complementary domain is a domain including a nucleic acid sequence complementary to the second complementary domain, and having enough complementarity so as to form a double strand with the second complementary domain.

Here, the first complementary domain may be or include a 5 to 35-base sequence. For example, the first complementary domain may be or include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

The first complementary domain may have homology with a natural first complementary domain, or may be derived from a natural first complementary domain. In addition, the first complementary domain may have a difference in the base sequence of a first complementary domain depending on the species existing in nature, may be derived from a first complementary domain contained in the species existing in nature, or may have partial or complete homology with the first complementary domain contained in the species existing in nature.

In one exemplary embodiment, the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium eligens, or a first complementary domain derived therefrom.

Selectively, the first complementary domain may include an additional base sequence which does not undergo complementary bonding with the second complementary domain.

Here, the additional base sequence may be a 1 to 15-base sequence. For example, the additional base sequence may be a 1 to 5, 5 to 10, or 10 to 15-base sequence.

Second Complementary Domain

The second complementary domain includes a nucleic acid sequence complementary to the first complementary domain, and has enough complementarity so as to form a double strand with the first complementary domain. The second complementary domain may include a base sequence complementary to the first complementary domain, and a base sequence having no complementarity with the first complementary domain, for example, a base sequence not forming a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain.

Here, the second complementary domain may be or include a 5 to 35-base sequence. For example, the second complementary domain may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

The second complementary domain may have homology with a natural second complementary domain, or may be derived from the natural second complementary domain. In addition, the second complementary domain may have a difference in base sequence of the second complementary domain according to a species existing in nature, and may be derived from second complementary domain contained in the species existing in nature, or may have partial or complete homology with the second complementary domain contained in the species existing in nature.

In one exemplary embodiment, the second complementary domain may have partial, that is, at least 50% or more, or complete homology with a second complementary domain of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium eligens, or a second complementary domain derived therefrom.

Selectively, the second complementary domain may include an additional base sequence which does not undergo complementary bonding with the first complementary domain.

Here, the additional base sequence may be a 1 to 15-base sequence. For example, the additional base sequence may be a 1 to 5, 5 to 10, or 10 to 15-base sequence.

Linker Domain

Selectively, the linker domain is a nucleic acid sequence connecting a first complementary domain with a second complementary domain to produce single-stranded gRNA. Here, the linker domain may be connected with the first complementary domain and the second complementary domain, or may connect the first and second complementary domains by covalent or non-covalent bonding.

The linker domain may be or include a 1 to 30-base sequence. For example, the linker domain may be or include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25 or 25 to 30-base sequence.

Selectively, a part or all of the base sequence of the guide domain, the first complementary domain, the second complementary domain and the linker domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.

Therefore, the single-stranded gRNA may consist of 5′-[second complementary domain]-[first complementary domain]-[guide domain]-3′ or 5′-[second complementary domain]-[linker domain]-[first complementary domain]-[guide domain]-3′ as described above.

In addition, the single-stranded gRNA may selectively include an additional base sequence.

In one exemplary embodiment, the single-stranded gRNA may be 5-(Z)_(h)-(Q)_(m)-(N_(target))-3′; or 5′-(X)_(a)-(Z)_(h)-(X)_(b)-(Q)_(m)-(X)_(c)-(N_(target))-3′. In another embodiment, the single-stranded gRNA may be 5′-(Z)_(h)-(L)_(j)-(Q)_(m)-(N_(target))-3′; or 5-(X)_(a)-(Z)_(h)-(L)_(j)-(Q)_(m)-(X)_(c)-(N_(target))-3′.

Here, the N_(target) is a base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, and a base sequence region which may be changed according to a target sequence on a target gene or nucleic acid.

In one exemplary embodiment, Ntarget may be a base sequence capable of forming a complementary bond with a target gene, that is, a target sequence of a neovascularization-associated factor such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene or an ANGPTL4 gene.

The (Q)m is a base sequence including the first complementary domain, which is able to form a complementary bond with the second complementary domain of the second strand. The (Q)m may be a sequence having partial or complete homology with the first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin. The Q may be each independently selected from the group consisting of A, U, C and G, and the m may be the number of bases, which is an integer of 5 to 35.

For example, when the first complementary domain has partial or complete homology with a first complementary domain of Parcubacteria bacterium or a first complementary domain derived therefrom, the (Q)m may be 5′-UUUGUAGAU-3′, or a base sequence having at least 50% or more homology with 5′-UUUGUAGAU-3′.

The (Z)h is a base sequence including a second complementary domain, which is able to form a complementary bond with the first complementary domain of the first strand. The (Z)h may be a sequence having partial or complete homology with the second complementary domain of a species existing in nature, and the base sequence of the second complementary domain may be modified according to the species of origin. The Z may be each independently selected from the group consisting of A, U, C and G, and the h may be the number of bases, which is an integer of 5 to 50.

For example, when the second complementary domain has partial or complete homology with a second complementary domain of Parcubacteria bacterium or a Parcubacteria bacterium-derived second complementary domain, the (Z)h may be 5′-AAAUUUCUACU-3′, or a base sequence having at least 50% or more homology with 5′-AAAUUUCUACU-3′.

In addition, the (L)j is a base sequence including the linker domain, which connects the first complementary domain with the second complementary domain. Here, the L may be each independently selected from the group consisting of A, U, C and G, and the j may be the number of bases, which is an integer of 1 to 30.

In addition, each of the (X)a, (X)b and (X)c is selectively an additional base sequence, where the X may be each independently selected from the group consisting of A, U, C and G, and the a, b and c may be the number of bases, which is 0 or an integer of 1 to 20.

2. Editor Protein

An editor protein refers to a peptide, polypeptide or protein which is able to directly bind to or interact with, without direct binding to, a nucleic acid.

The nucleic acid may be a nucleic acid contained in a target nucleic acid, gene or chromosome.

The nucleic acid may be a guide nucleic acid.

The editor protein may be an enzyme.

The editor protein may be a fusion protein.

Here, the fusion protein refers to a protein produced by fusing an enzyme with an additional domain, peptide, polypeptide or protein.

The enzyme refers to a protein including a domain which is able to cleave a nucleic acid, gene, chromosome or protein.

The enzyme may be a nuclease, protease or restriction enzyme.

The additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein, which has a function the same as or different from the enzyme.

The fusion protein may include an additional domain, peptide, polypeptide or protein at one or more of an N-terminus of an enzyme or the proximity thereof; a C-terminus of the enzyme or the proximity thereof; the middle region of an enzyme; and a combination thereof.

The fusion protein may include a functional domain, peptide, polypeptide or protein at one or more of an N-terminus of an enzyme or the proximity thereof; a C-terminus of the enzyme or the proximity thereof; the middle region of an enzyme; and a combination thereof.

Here, the functional domain, peptide, polypeptide or protein may be a domain, peptide, polypeptide or protein having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for isolation and purification of a protein (including a peptide), but the present invention is not limited thereto.

The functional domain, peptide, polypeptide or protein may be a deaminase.

The tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag and a thioredoxin (Trx) tag, and the reporter gene includes glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucoronidase, luciferase, autofluorescent proteins including the green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP), but the present invention is not limited thereto.

In addition, the functional domain, peptide, polypeptide or protein may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

The NLS may be NLS of SV40 virus large T-antigen with an amino acid sequence PKKKRKV; NLS derived from nucleoplasmin (e.g., nucleoplasmin bipartite NLS with a sequence KRPAATKKAGQAKKKK); c-myc NLS with an amino acid sequence PAAKRVKLD or RQRRNELKRSP; hRNPA1 M9 NLS with a sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; an importin-α-derived IBB domain sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; myoma T protein sequences VSRKRPRP and PPKKARED; human p53 sequence POPKKKPL; a mouse c-abl IV sequence SALIKKKKKMAP; influenza virus NS1 sequences DRLRR and PKQKKRK; a hepatitis virus-δ antigen sequence RKLKKKIKKL; a mouse Mx1 protein sequence REKKKFLKRR; a human poly(ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK; or steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK, but the present invention is not limited thereto.

The editor protein may include a complete active enzyme.

Here, the “complete active enzyme” refers to an enzyme having the same function as a function of a wild-type enzyme, and for example, the wild-type enzyme cleaving the double strand of DNA has complete enzyme activity of entirely cleaving the double strand of DNA.

In addition, the complete active enzyme includes an enzyme having an improved function compared to the function of the wild-type enzyme, and for example, a specific modification or manipulation type of the wild-type enzyme cleaving the double strand of DNA has full enzyme activity which is improved compared to the wild-type enzyme, that is, activity of cleaving the double strand of DNA.

The editor protein may include an incomplete or partially active enzyme.

Here, the “incomplete or partially active enzyme” refers to an enzyme having some of the functions of the wild-type enzyme, and for example, a specific modification or manipulation type of the wild-type enzyme cleaving the double strand of DNA has incomplete or partial enzyme activity of cleaving a part of the double strand, that is, a single strand of DNA.

The editor protein may include an inactive enzyme.

Here, the “inactive enzyme” refers to an enzyme in which the function of a wild-type enzyme is completely inactivated. For example, a specific modification or manipulation type of the wild-type enzyme cleaving the double strand of DNA has inactivity so as not to completely cleave the DNA double strand.

The editor protein may be a natural enzyme or fusion protein.

The editor protein may be present in the form of a partially modified natural enzyme or fusion protein.

The editor protein may be an artificially produced enzyme or fusion protein, which does not exist in nature.

The editor protein may be present in the form of a partially modified artificial enzyme or fusion protein, which does not exist in nature.

Here, the modification may be substitution, removal, addition of amino acids contained in the editor protein, or a combination thereof.

In addition, the modification may be substitution, removal, addition of some bases in the base sequence encoding the editor protein, or a combination thereof.

As one exemplary embodiment of the editor protein of the present invention, a CRISPR enzyme will be described below.

CRISPR Enzyme

The term “CRISPR enzyme” is a main protein component of a CRISPR-Cas system, and forms a complex with gRNA, resulting in the CRISPR-Cas system.

The CRISPR enzyme is a nucleic acid or polypeptide (or a protein) having a sequence encoding the CRISPR enzyme, and representatively, a Type II CRISPR enzyme or Type V CRISPR enzyme is widely used.

The Type II CRISPR enzyme is Cas9, which may be derived from various microorganisms such as Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus and Acaryochloris marina.

The term “Cas9” is an enzyme which binds to gRNA so as to cleave or modify a target sequence or position on a target gene or nucleic acid, and may consist of an HNH domain capable of cleaving a nucleic acid strand forming a complementary bond with gRNA, an RuvC domain capable of cleaving a nucleic acid strand forming a complementary bond with gRNA, an REC domain recognizing a target and a PI domain recognizing PAM. Hiroshi Nishimasu et al. (2014) Cell 156:935-949 may be referenced for specific structural characteristics of Cas9.

In addition, the Type V CRISPR enzyme may be Cpf1, which may be derived from Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Methylobacterium or Acidaminococcus.

The Cpf1 may consist of an RuvC domain similar and corresponding to the RuvC domain of Cas9, an Nuc domain without the HNH domain of Cas9, an REC domain recognizing a target, a WED domain and a PI domain recognizing PAM. For specific structural characteristics of Cpf1, Takashi Yamano et al. (2016) Cell 165:949-962 may be referenced.

The CRISPR enzyme of the Cas9 or Cpf1 protein may be isolated from a microorganism existing in nature or non-naturally produced by a recombinant or synthetic method.

Type II CRISPR Enzyme

The crystal structure of the type II CRISPR enzyme was determined according to studies on two or more types of natural microbial type II CRISPR enzyme molecules (Jinek et al., Science, 343(6176):1247997, 2014) and studies on Streptococcus pyogenes Cas9 (SpCas9) complexed with gRNA (Nishimasu et al., Cell, 156:935-949, 2014; and Anders et al., Nature, 2014, doi: 10.1038/nature13579).

The type II CRISPR enzyme includes two lobes, that is, recognition (REC) and nuclease (NUC) lobes, and each lobe includes several domains.

The REC lobe includes an arginine-rich bridge helix (BH) domain, an REC1 domain and an REC2 domain.

Here, the BH domain is a long α-helix and arginine-rich region, and the REC1 and REC2 domains play an important role in recognizing a double strand formed in gRNA, for example, single-stranded gRNA, double-stranded gRNA or tracrRNA.

The NUC lobe includes an RuvC domain, an HNH domain and a PAM-interaction (PI) domain. Here, the RuvC domain encompasses RuvC-like domains, or the HNH domain is used to include HNH-like domains.

Here, the RuvC domain shares structural similarity with members of the microorganism family existing in nature having the type II CRISPR enzyme, and cleaves a single strand, for example, a non-complementary strand of a target gene or nucleic acid, that is, a strand not forming a complementary bond with gRNA. The RuvC domain is sometimes referred to as an RuvCI domain, RuvCII domain or RuvCIII domain in the art, and generally called an RuvC I, RuvCII or RuvCIII. For example, in the case of SpCas9, the RuvC domain is assembled from each of three divided RuvC domains (RuvC I, RuvCII and RuvCIII) located at the sequences of amino acids 1 to 59, 718 to 769 and 909 to 1098 of SpCas9, respectively.

The HNH domain shares structural similarity with the HNH endonuclease, and cleaves a single strand, for example, a complementary strand of a target nucleic acid molecule, that is, a strand forming a complementary bond with gRNA. The HNH domain is located between RuvC II and III motifs. For example, in the case of SpCas9, the HNH domain is located at amino acid sequence 775 to 908 of SpCas9.

The PI domain recognizes a specific base sequence in a target gene or nucleic acid, that is, a protospacer adjacent motif (PAM) or interacts with PAM. For example, in the case of SpCas9, the PI domain is located at the sequence of amino acids1099 to 1368 of SpCas9.

Here, the PAM may vary according to the origin of the type II CRISPR enzyme. For example, when the CRISPR enzyme is SpCas9, PAM may be 5′-NGG-3′, when the CRISPR enzyme is Streptococcus thermophilus Cas9 (StCas9), PAM may be 5′-NNAGAAW-3′(W=A or T), when the CRISPR enzyme is Neisseria meningitides Cas9 (NmCas9), PAM may be 5′-NNNNGATT-3′, and when the CRISPR enzyme is Campylobacter jejuni Cas9 (CjCas9), PAM may be 5′-NNNVRYAC-3′ (V=G or C or A, R=A or G, Y=C or T), where the N may be A, T, G or C; or A, U, G or C.

Type V CRISPR Enzyme

Type V CRISPR enzyme includes similar RuvC domains corresponding to the RuvC domains of the type II CRISPR enzyme, and may consist of an Nuc domain, instead of the HNH domain of the type II CRISPR enzyme, REC and WED domains, which recognize a target, and a PI domain recognizing PAM. For specific structural characteristics of the type V CRISPR enzyme, Takashi Yamano et al. (2016) Cell 165:949-962 may be referenced.

The type V CRISPR enzyme may interact with gRNA, thereby forming a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, and may allow a guide sequence to approach a target sequence including a PAM sequence in cooperation with gRNA. Here, the ability of the type V CRISPR enzyme for interaction with a target gene or nucleic acid is dependent on the PAM sequence.

The PAM sequence is a sequence present in a target gene or nucleic acid, and may be recognized by the PI domain of the type V CRISPR enzyme. The PAM sequence may vary according to the origin of the type V CRISPR enzyme. That is, there are different PAM sequences which are able to be specifically recognized depending on a species.

In one example, the PAM sequence recognized by Cpf1 may be 5′-TTN-3′ (N is A, T, C or G).

CRISPR Enzyme Activity

A CRISPR enzyme cleaves a double or single strand of a target gene or nucleic acid, and has nuclease activity causing breakage or deletion of the double or single strand. Generally, the wild-type type II CRISPR enzyme or type V CRISPR enzyme cleaves the double strand of the target gene or nucleic acid.

To manipulate or modify the above-described nuclease activity of the CRISPR enzyme, the CRISPR enzyme may be manipulated or modified, such a manipulated or modified CRISPR enzyme may be modified into an incompletely or partially active or inactive enzyme.

Incompletely or Partially Active Enzyme

A CRISPR enzyme modified to change enzyme activity, thereby exhibiting incomplete or partial activity is called a nickase.

The term “nickase” refers to a CRISPR enzyme manipulated or modified to cleave only one strand of the double strand of the target gene or nucleic acid, and the nickase has nuclease activity of cleaving a single strand, for example, a strand that is not complementary or complementary to gRNA of the target gene or nucleic acid. Therefore, to cleave the double strand, nuclease activity of the two nickases is needed.

For example, the nickase may have nuclease activity by the RuvC domain. That is, the nickase may include nuclease activity of the HNH domain, and to this end, the HNH domain may be manipulated or modified.

In one example, provided that the CRISPR enzyme is the type II CRISPR enzyme, when the residue 840 in the amino acid sequence of SpCas9 is mutated from histidine to alanine, the nuclease activity of the HNH domain is inactivated to be used as a nickase. Since the nickase produced thereby has nuclease activity of the RuvC domain, it is able to cleave a strand which does not form a complementary bond with a non-complementary strand of the target gene or nucleic acid, that is, gRNA.

In another exemplary embodiment, when the residue 559 in the amino acid sequence of CjCas9 is mutated from histidine to alanine, the nuclease activity of the HNH domain is inactivated to be used as a nickase. The nickase produced thereby has nuclease activity by the RuvC domain, and thus is able to cleave a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with gRNA.

For example, the nickase may have nuclease activity by the HNH domain. That is, the nickase may include the nuclease activity of the RuvC domain, and to this end, the RuvC domain may be manipulated or modified.

In one example, provided that the CRISPR enzyme is the type II CRISPR enzyme, in one exemplary embodiment, when the residue 10 in the amino acid sequence of SpCas9 is mutated from aspartic acid to alanine, the nuclease activity of the RuvC domain is inactivated to be used as a nickase. The nickase produced thereby has the nuclease activity of the HNH domain, and thus is able to cleave a complementary strand of the target gene or nucleic acid, that is, a strand that forms a complementary bond with gRNA.

In another exemplary embodiment, when the residue 8 in the amino acid sequence of CjCas9 is mutated from aspartic acid to alanine, the nuclease activity of the RuvC domain is inactivated to be used as a nickase. The nickase produced thereby has the nuclease activity of the HNH domain, and thus is able to cleave a complementary strand of the target gene or nucleic acid, that is, a strand that forms a complementary bond with gRNA.

Inactive Enzyme

A CRISPR enzyme which is modified to make enzyme activity completely inactive is called an inactive CRISPR enzyme.

The term “inactive CRISPR enzyme” refers to a CRISPR enzyme which is modified not to completely cleave the double strand of the target gene or nucleic acid, and the inactive CRISPR enzyme has nuclease inactivity due to the mutation in the domain with nuclease activity of the wild-type CRISPR enzyme. The inactive CRISPR enzyme may be one in which the nuclease activities of the RuvC domain and the HNH domain are inactivated.

For example, the inactive CRISPR enzyme may be manipulated or modified in the RuvC domain and the HNH domain so as to inactive nuclease activity.

In one example, provided that the CRISPR enzyme is the type II CRISPR enzyme, in one exemplary embodiment, when the residues 10 and 840 in the amino acid sequence of SpCas9 are mutated from aspartic acid and histidine to alanine, respectively, nuclease activities by the RuvC domain and the HNH domain are inactivated, such that the double strand may not cleave completely the double strand of the target gene or nucleic acid.

In another exemplary embodiment, when the residues 8 and 559 in the amino acid sequence of CjCas9 are mutated from aspartic acid and histidine to alanine, the nuclease activities by the RuvC domain and the HNH domain are inactivated, such that the double strand may not cleave completely the double strand of the target gene or nucleic acid.

Other Activities

The CRISPR enzyme may have endonuclease activity, exonuclease activity or helicase activity, that is, an ability to anneal the helix structure of the double-stranded nucleic acid, in addition to the above-described nuclease activity.

In addition, the CRISPR enzyme may be modified to completely, incompletely, or partially activate the endonuclease activity, exonuclease activity or helicase activity.

Targeting of CRISPR Enzyme

The CRISPR enzyme may interact with gRNA, thereby forming a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, and lead a guide sequence to approach a target sequence including a PAM sequence in cooperation with gRNA. Here, the ability of the CRISPR enzyme to interact with the target gene or nucleic acid is dependent on the PAM sequence.

The PAM sequence is a sequence present in the target gene or nucleic acid, which may be recognized by the PI domain of the CRISPR enzyme. The PAM sequence may vary depending on the origin of the CRISPR enzyme. That is, there are various PAM sequences which are able to be specifically recognized according to species.

In one example, provided that the CRISPR enzyme is the type II CRISPR enzyme,

-   -   in the case of SpCas9, the PAM sequence may be 5′-NGG-3′,         5′-NAG-3′ and/or 5′-NGA-3′,     -   in the case of StCas9, the PAM sequence may be 5′-NGGNG-3′         and/or 5′-NNAGAAW-3′ (W=A or T),     -   in the case of NmCas9, the PAM sequence may be 5′-NNNNGATT-3′         and/or 5′-NNNGCTT-3′,     -   in the case of CjCas9, the PAM sequence may be 5′-NNNVRYAC-3′         (V=G, C or A; R=A or G; Y=C or T),     -   in the case of Streptococcus mutans Cas9 (SmCas9), the PAM         sequence may be 5′-NGG-3′ and/or 5′-NAAR-3′ (R=A or G), and     -   in the case of Staphylococcus aureus Cas9 (SaCas9), the PAM         sequence may be 5′-NNGRR-3′, 5′-NNGRRT-3′ and/or 5′-NNGRRV-3′         (R=A or G; V=G, C or A).

In another example, provided that the CRISPR enzyme is the type V CRISPR enzyme, in the case of Cpf1, the PAM sequence may be 5′-TTN-3′.

Here, the N may be A, T, G or C; or A, U, G or C.

The CRISPR enzyme capable of recognizing a specific PAM sequence may be manipulated or modified using the PAM sequence capable of being specifically recognized according to species. For example, the PI domain of SpCas9 may be replaced with the PI domain of CjCas9 so as to have the nuclease activity of SpCas9 and recognize a CjCas9-specific PAM sequence, thereby producing SpCas9 recognizing the CjCas9-specific PAM sequence. A specifically recognized PAM sequence may be changed by substitution or replacement of the PI domain.

CRISPR Enzyme Mutant

The CRISPR enzyme may be modified to improve or inhibit various characteristics such as nuclease activity, helicase activity, an ability to interact with gRNA, and an ability to approach the target gene or nucleic acid, for example, PAM recognizing ability of the CRISPR enzyme.

In addition, the CRISPR enzyme mutant may be a CRISPR enzyme which interacts with gRNA to form a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, and is modified or manipulated to improve target specificity, when approaching or localized to the target gene or nucleic acid, such that only a double or single strand of the target gene or nucleic acid is cleaved without cleavage of a double or single strand of a non-target gene or nucleic acid which partially forms a complementary bond with gRNA and a non-target gene or nucleic acid which does not form a complementary bond therewith.

Here, an effect of cleaving the double or single strand of the non-target gene or nucleic acid partially forming a complementary bond with gRNA and the non-target gene or nucleic acid not forming a complementary bond therewith is referred to as an off-target effect, a position or base sequence of the non-target gene or nucleic acid partially forming a complementary bond with gRNA and the non-target gene or nucleic acid not forming a complementary bond therewith is referred to as an off-target. Here, there may be one or more off-targets. One the other hand, the cleavage effect of the double or single strand of the target gene or nucleic acid is referred to as an on-target effect, and a location or target sequence of the target gene or nucleic acid is referred to as an on-target.

The CRISPR enzyme mutant is modified in at least one of the amino acids of a naturally-occurring CRISPR enzyme, and may be modified, for example, improved or inhibited in one or more of the various characteristics such as nuclease activity, helicase activity, an ability to interact with gRNA, an ability to approach the target gene or nucleic acid and target specificity, compared to the unmodified CRISPR enzyme. Here, the modification may be substitution, removal, addition of an amino acid, or a mixture thereof.

In the CRISPR enzyme mutant, the modification may be a modification of one or two or more amino acids located in a region consisting of amino acids having positive charges, present in the naturally-occurring CRISPR enzyme.

For example, the modification may be a modification of one or two or more amino acids of the positively-charged amino acids such as lysine (K), arginine (R) and histidine (H), present in the naturally-occurring CRISPR enzyme.

The modification may be a modification of one or two or more amino acids located in a region composed of non-positively-charged amino acids present in the naturally-occurring CRISPR enzyme.

For example, the modification may be a modification of one or two or more amino acids of the non-positively-charged amino acids, that is, aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.

In another example, the modification may be a modification of one or two or more amino acids of non-charged amino acids, that is, serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.

In addition, the modification may be a modification of one or two or more of the amino acids having hydrophobic residues present in the naturally-occurring CRISPR enzyme.

For example, the modification may be a modification of one or two or more amino acids of glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.

The modification may be a modification of one or two or more of the amino acids having polar residues, present in the naturally-occurring CRISPR enzyme.

For example, the modification may be a modification of one or two or more amino acids of serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), lysine (K), arginine (R), histidine (H), aspartic acid (D) and glutamic acid (E), present in the naturally-occurring CRISPR enzyme.

In addition, the modification may be a modification of one or two or more of the amino acids including lysine (K), arginine (R) and histidine (H), present in the naturally-occurring CRISPR enzyme.

For example, the modification may be a substitution of one or two or more of the amino acids including lysine (K), arginine (R) and histidine (H), present in the naturally-occurring CRISPR enzyme.

The modification may be a modification of one or two or more of the amino acids including aspartic acid (D) and glutamic acid (E), present in the naturally-occurring CRISPR enzyme.

For example, the modification may be a substitution of one or two or more of the amino acids including aspartic acid (D) and glutamic acid (E), present in the naturally-occurring CRISPR enzyme.

The modification may be a modification of one or two or more of the amino acids including serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.

For example, the modification may be a substitution of one or two or more of the amino acid including serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.

In addition, the modification may be a modification of one, two, three, four, five, six, seven or more of the amino acids present in the naturally-occurring CRISPR enzyme.

In addition, in the CRISPR enzyme mutant, the modification may be a modification of one or two or more of the amino acids present in the RuvC domain of the CRISPR enzyme. Here, the RuvC domain may be an RuvCI, RuvCII or RuvCIII domain.

The modification may be a modification of one or two or more of the amino acids present in the HNH domain of the CRISPR enzyme.

The modification may be a modification of one or two or more of the amino acids present in the REC domain of the CRISPR enzyme.

The modification may be one or two or more of the amino acids present in the PI domain of the CRISPR enzyme.

The modification may be a modification of two or more of the amino acids contained in at least two or more domains of the REC, RuvC, HNH and PI domains of the CRISPR enzyme.

In one example, the modification may be a modification of two or more of the amino acids contained in the REC and RuvC domains of the CRISPR enzyme.

In one exemplary embodiment, in the SpCas9 mutant, the modification may be a modification of at least two or more of the A203, H277, G366, F539, I601, M763, D965 and F1038 amino acids contained in the REC and RuvC domains of SpCas9.

In another example, the modification may be a modification of two or more of the amino acids contained in the REC and HNH domains of the CRISPR enzyme.

In one exemplary embodiment, in the SpCas9 mutant, the modification may be a modification of at least two or more of the A203, H277, G366, F539, I601 and K890 amino acids contained in the REC and HNH domains of SpCas9.

In one example, the modification may be a modification of two or more of the amino acids contained in the REC and PI domains of the CRISPR enzyme.

In one exemplary embodiment, in the SpCas9 mutant, the modification may be a modification of at least two or more of the A203, H277, G366, F539, I601, T1102 and D1127 amino acids contained in the REC and PI domains of SpCas9.

In another example, the modification may be a modification of three or more of the amino acids contained in the REC, RuvC and HNH domains of the CRISPR enzyme.

In one exemplary embodiment, in the SpCas9 mutant, the modification may be a modification of at least three or more of the A203, H277, G366, F539, I601, M763, K890, D965 and F1038 amino acids contained in the REC, RuvC and HNH domains of SpCas9.

In one example, the modification may be a modification of three or more of the amino acids contained in the REC, RuvC and PI domains contained in the CRISPR enzyme.

In one exemplary embodiment, in the SpCas9 mutant, the modification may be a modification of at least three or more of the A203, H277, G366, F539, I601, M763, D965, F1038, T1102 and D1127 amino acids contained in the REC, RuvC and PI domains of SpCas9.

In another example, the modification may be a modification of three or more of the amino acids contained in the REC, HNH and PI domains of the CRISPR enzyme.

In one exemplary embodiment, in the SpCas9 mutant, the modification may be a modification of at least three or more of the A203, H277, G366, F539, I601, K890, T1102 and D1127 amino acids contained in the REC, HNH and PI domains of SpCas9.

In one example, the modification may be a modification of three or more of the amino acids contained in the RuvC, HNH and PI domains of the CRISPR enzyme.

In one exemplary embodiment, in the SpCas9 mutant, the modification may be a modification of at least three or more of the M763, K890, D965, F1038, T1102 and D1127 amino acids contained in the RuvC, HNH and PI domains of SpCas9.

In another example, the modification may be a modification of four or more of the amino acids contained in the REC, RuvC, HNH and PI domains of the CRISPR enzyme.

In one exemplary embodiment, in the SpCas9 mutant, the modification may be a modification of at least four or more of the A203, H277, G366, F539, I601, M763, K890, D965, F1038, T1102 and D1127 amino acids contained in the REC, RuvC, HNH and PI domains of SpCas9.

In addition, in the CRISPR enzyme mutant,

the modification may be a modification of one or two or more of the amino acids participating in the nuclease activity of the CRISPR enzyme.

For example, in the SpCas9 mutant, the modification may be a modification of one or two or more of the group consisting of the amino acids D10, E762, H840, N854, N863 and D986, or one or two or more of the group consisting of the amino acids corresponding to other Cas9 orthologs.

The modification may be a modification for partially inactivating the nuclease activity of the CRISPR enzyme, and such a CRISPR enzyme mutant may be a nickase.

Here, the modification may be a modification for inactivating the nuclease activity of the RuvC domain of the CRISPR enzyme, and such a CRISPR enzyme mutant may not cleave a non-complementary strand of a target gene or nucleic acid, that is, a strand which does not form a complementary bond with gRNA.

In one exemplary embodiment, in the case of SpCas9, when residue 10 of the amino acid sequence of SpCas9 is mutated from aspartic acid to alanine, that is, when mutated to D10A, the nuclease activity of the RuvC domain is inactivated, and thus the SpCas9 may be used as a nickase. The nickase produced thereby may not cleave a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with gRNA.

In another exemplary embodiment, in the case of CjCas9, when residue 8 of the amino acid sequence of CjCas9 is mutated from aspartic acid to alanine, that is, when mutated to D8A, the nuclease activity of the RuvC domain is inactivated, and thus the CjCas9 may be used as a nickase. The nickase produced thereby may not cleave a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with gRNA.

In addition, here, the modification may be a modification for inactivating the nuclease activity of the HNH domain of the CRISPR enzyme, and such a CRISPR enzyme mutant may not cleave a complementary strand of the target gene or nucleic acid, that is, a strand forming a complementary bond with gRNA.

In one exemplary embodiment, in the case of SpCas9, when residue 840 of the amino acid sequence of SpCas9 is mutated from histidine to alanine, that is, when mutated to H840A, the nuclease activity of the HNH domain is inactivated, and thus the SpCas9 may be used as a nickase. The nickase produced thereby may not cleave a complementary strand of the target gene or nucleic acid, that is, a strand that forms a complementary bond with gRNA.

In another exemplary embodiment, in the case of CjCas9, when residue 559 of the amino acid sequence of CjCas9 is mutated from histidine to alanine, that is, when mutated to H559A, the nuclease activity of the HNH domain is inactivated, and thus the CjCas9 may be used as a nickase. The nickase produced thereby may not cleave a complementary strand of the target gene or nucleic acid, that is, a strand that forms a complementary bond with g RNA.

In addition, the modification may be a modification for completely inactivating the nuclease activity of the CRISPR enzyme, and such a CRISPR enzyme mutant may be an inactive CRISPR enzyme.

Here, the modification may be a modification for inactivating the nuclease activities of the RuvC and HNH domains of the CRISPR enzyme, and such a CRISPR enzyme mutant may does not cleave a double strand of the target gene or nucleic acid.

In one exemplary embodiment, in the case of SpCas9, when the residues 10 and 840 in the amino acid sequence of SpCas9 are mutated from aspartic acid and histidine to alanine, that is, mutated to D10A and H840A, respectively, the nuclease activities of the RuvC domain and the HNH domain are inactivated, the double strand of the target gene or nucleic acid may not be completely cleaved.

In another exemplary embodiment, in the case of CjCas9, when residues 8 and 559 of the amino acid sequence of CjCas9 are mutated from aspartic acid and histidine to alanine, that is, mutated to D8A and H559A, respectively, the nuclease activities by the RuvC and HNH domains are inactivated, and thus the double strand of the target gene or nucleic acid may not be completely cleaved.

In addition, the CRISPR enzyme mutant may further include an optionally functional domain, in addition to the innate characteristics of the CRISPR enzyme, and such a CRISPR enzyme mutant may have an additional characteristic in addition to the innate characteristics.

Here, the functional domain may be a domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for isolating and purifying a protein (including a peptide), but the present invention is not limited thereto.

The functional domain, peptide, polypeptide or protein may be a deaminase.

For example, an incomplete or partial CRISPR enzyme may additionally include a cytidine deaminase as a functional domain. In one exemplary embodiment, a cytidine deaminase, for example, apolipoprotein B editing complex 1 (APOBEC1) may be added to SpCas9 nickase, thereby producing a fusion protein. The [SpCas9 nickase]-[APOBEC1] formed thereby may be used in base repair or editing of C into T or U, or G into A.

The tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag and a thioredoxin (Trx) tag, and the reporter gene includes glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucoronidase, luciferase, autofluorescent proteins including the green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP), but the present invention is not limited thereto.

In addition, the functional domain may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

In one example, the CRISPR enzyme may include one or more NLSs. Here, one or more NLSs may be included at an N-terminus of an CRISPR enzyme or the proximity thereof; a C-terminus of the enzyme or the proximity thereof; or a combination thereof. The NLS may be an NLS sequence derived from the following NLSs, but the present invention is not limited thereto: NLS of a SV40 virus large T-antigen having the amino acid sequence PKKKRKV; NLS from nucleoplasmin (e.g., nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-α; the sequences VSRKRPRP and PPKKARED of a myoma T protein; the sequence POPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-abl IV; the sequences DRLRR and PKQKKRK of influenza virus NS1; the sequence RKLKKKIKKL of a hepatitis delta virus antigen; the sequence REKKKFLKRR of a mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK of a human poly (ADP-ribose) polymerase; or the NLS sequence RKCLQAGMNLEARKTKK, derived from a sequence of a steroid hormone receptor (human) glucocorticoid.

In addition, the CRISPR enzyme mutant may include a split-type CRISPR enzyme prepared by dividing the CRISPR enzyme into two or more parts. The term “split” refers to functional or structural division of a protein or random division of a protein into two or more parts.

Here, the split-type CRISPR enzyme may be a completely, incompletely or partially active enzyme or inactive enzyme.

For example, the SpCas9 may be divided into two parts between the residue 656, tyrosine, and the residue 657, threonine, thereby generating split SpCas9.

In addition, the split-type CRISPR enzyme may selectively include an additional domain, peptide, polypeptide or protein for reconstitution.

Here, the “reconstitution” refers to formation of the split-type CRISPR enzyme to be structurally the same or similar to the wild-type CRISPR enzyme.

The additional domain, peptide, polypeptide or protein for reconstitution may be FRB and FKBP dimerization domains; intein; ERT and VPR domains; or domains which form a heterodimer under specific conditions.

For example, the SpCas9 may be divided into two parts between the residue 713, serine, and the residue 714, glycine, thereby generating split SpCas9. The FRB domain may be connected to one of the two parts, and the FKBP domain may be connected to the other one. In the split SpCas9 produced thereby, the FRB domain and the FKBP domain may be formed in a dimer in an environment in which rapamycine is present, thereby producing a reconstituted CRISPR enzyme.

The CRISPR enzyme or CRISPR enzyme mutant described in the present invention may be a polypeptide, protein or nucleic acid having a sequence encoding the same, and may be codon-optimized for a subject to introduce the CRISPR enzyme or CRISPR enzyme mutant.

The term “codon optimization” refers to a process of modifying a nucleic acid sequence by maintaining a native amino acid sequence while replacing at least one codon of the native sequence with a codon more frequently or the most frequently used in host cells so as to improve expression in the host cells. A variety of species have a specific bias to a specific codon of a specific amino acid, and the codon bias (the difference in codon usage between organisms) is frequently correlated with efficiency of the translation of mRNA, which is considered to be dependent on the characteristic of a translated codon and availability of a specific tRNA molecule. The dominance of tRNA selected in cells generally reflects codons most frequently used in peptide synthesis. Therefore, a gene may be customized by optimal gene expression in a given organism based on codon optimization.

3. Target Sequence

The term “target sequence” is a base sequence present in a target gene or nucleic acid, and has complementarity to a guide sequence contained in a guide domain of a guide nucleic acid. The target sequence is a base sequence which may vary according to a target gene or nucleic acid, that is, a subject for gene manipulation or correction, which may be designed in various forms according to the target gene or nucleic acid.

The target sequence may form a complementary bond with the guide sequence contained in the guide domain of the guide nucleic acid, and a length of the target sequence may be the same as that of the guide sequence.

The target sequence may be a 5 to 50-base sequence.

In an embodiment, the target sequence may be a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.

The target sequence may be a nucleic acid sequence complementary to the guide sequence contained in the guide domain of the guide nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

In one example, the target sequence may be or include a 1 to 8-base sequence, which is not complementary to the guide sequence contained in the guide domain of the guide nucleic acid.

In addition, the target sequence may be a base sequence adjacent to a nucleic acid sequence that is able to be recognized by an editor protein.

In one example, the target sequence may be a continuous 5 to 50-base sequence adjacent to the 5′ end and/or 3′ end of the nucleic acid sequence that is able to be recognized by the editor protein.

In one exemplary embodiment, target sequences for a gRNA-CRISPR enzyme complex will be described below.

When the target gene or nucleic acid is targeted by the gRNA-CRISPR enzyme complex, the target sequence has complementarity to the guide sequence contained in the guide domain of gRNA. The target sequence is a base sequence which varies according to the target gene or nucleic acid, that is, a subject for gene manipulation or correction, which may be designed in various forms according to the target gene or nucleic acid.

In addition, the target sequence may be a base sequence adjacent to a PAM sequence which is able to be recognized by the CRISPR enzyme, that is, Cas9 or Cpf1.

In one example, the target sequence may be a continuous 5 to 50-base sequence adjacent to the 5′ end and/or 3′ end of the PAM sequence which is recognized by the CRISPR enzyme.

In one exemplary embodiment, when the CRISPR enzyme is SpCas9, the target sequence may be a continuous 16 to 25-base sequence adjacent to the 5′ end and/or 3′ end of a 5′-NGG-3′, 5′-NAG-3′ and/or 5′-NGA-3′ (N=A, T, G or C; or A, U, G or C) sequence.

In another exemplary embodiment, when the CRISPR enzyme is StCas9, the target sequence may be a continuous 16 to 25-base sequence adjacent to the 5′ end and/or 3′ end of a 5′-NGGNG-3′ and/or 5′-NNAGAAW-3′ (W=A or T, and N=A, T, G or C; or A, U, G or C) sequence.

In still another exemplary embodiment, when the CRISPR enzyme is NmCas9, the target sequence may be a continuous 16 to 25-base sequence adjacent to the 5′ end and/or 3′ end of a 5′-NNNNGATT-3′ and/or 5′-NNNGCTT-3′ (N=A, T, G or C; or A, U, G or C) sequence.

In one exemplary embodiment, when the CRISPR enzyme is CjCas9, the target sequence may be a continuous 16 to 25-base sequence adjacent to the 5′ end and/or 3′ end of a 5′-NNNVRYAC-3′ (V=G, C or A; R=A or G, Y=C or T, N=A, T, G or C; or A, U, G or C) sequence.

In another exemplary embodiment, when the CRISPR enzyme is SmCas9, the target sequence may be a continuous 16 to 25-base sequence adjacent to the 5′ end and/or 3′ end of a 5′-NGG-3′ and/or 5′-NAAR-3′(R=A or G, N=A, T, G or C; or A, U, G or C) sequence.

In yet another exemplary embodiment, when the CRISPR enzyme is SaCas9, the target sequence may be a continuous 16 to 25-base sequence adjacent to the 5′ end and/or 3′ end of a 5′-NNGRR-3′, 5′-NNGRRT-3′ and/or 5′-NNGRRV-3′ (R=A or G, V=G, C or A, N=A, T, G or C; or A, U, G or C) sequence.

In one exemplary embodiment, when the CRISPR enzyme is Cpf1, the target sequence may be a continuous 16 to 25-base sequence adjacent to the 5′ end and/or 3′ end of a 5′-TTN-3′ (N=A, T, G or C; or A, U, G or C) sequence.

In one exemplary embodiment of the present invention, the target sequence may be a nucleic acid sequence contained in one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.

The target sequence may be a nucleic acid sequence contained in the VEGFA gene.

The target sequence may be a nucleic acid sequence contained in the HIF1A gene.

The target sequence may be a nucleic acid sequence contained in the ANGPT2 gene.

The target sequence may be a nucleic acid sequence contained in the EPAS1 gene.

The target sequence may be a nucleic acid sequence contained in the ANGPTL4 gene.

Alternatively, the target sequence may be a partial nucleic acid sequence of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.

The target sequence may be a partial nucleic acid sequence of the VEGFA gene.

The target sequence may be a partial nucleic acid sequence of the HIF1A gene.

The target sequence may be a partial nucleic acid sequence of the ANGPT2 gene.

The target sequence may be a partial nucleic acid sequence of the EPAS1 gene.

The target sequence may be a partial nucleic acid sequence of the ANGPTL4 gene.

Alternatively, the target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.

The target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the VEGFA gene.

The target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the HIF1A gene.

The target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the ANGPT2 gene.

The target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the EPAS1 gene.

The target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the ANGPTL4 gene.

Alternatively, the target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.

The target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the VEGFA gene.

The target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the HIF1A gene.

The target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the ANGPT2 gene.

The target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the EPAS1 gene.

The target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the ANGPTL4 gene.

Alternatively, the target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.

The target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the VEGFA gene.

The target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the HIF1A gene.

The target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the ANGPT2 gene.

The target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the EPAS1 gene.

The target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the ANGPTL4 gene.

Alternatively, The target sequence may be a nucleic acid sequence including or adjacent to a mutated region (e.g., a region different from a wild-type gene) of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.

The target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the VEGFA gene.

The target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the HIF1A gene.

The target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the ANGPT2 gene.

The target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the EPAS1 gene.

The target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the ANGPTL4 gene.

Alternatively, the target sequence may be a continuous 5 to 50-nucleic acid sequence of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and an ANGPTL4 gene.

The target sequence may be a continuous 5 to 50-nucleic acid sequence of the VEGFA gene.

The target sequence may be a continuous 5 to 50-nucleic acid sequence of the HIF1A gene.

The target sequence may be a continuous 5 to 50-nucleic acid sequence of the ANGPT2 gene.

The target sequence may be a continuous 5 to 50-nucleic acid sequence of the EPAS1 gene.

The target sequence may be a continuous 5 to 50-nucleic acid sequence of the ANGPTL4 gene.

As one exemplary embodiment of the present invention, the above target sequences of the VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and ANGPTL4 gene are summarized in Table 1, Table 2, Table 3, Table 4 and Table 5.

Neovascularization-Associated Factor-Manipulated Product

4. Guide Nucleic Acid-Editor Protein Complex and Use Thereof

A guide nucleic acid-editor protein complex may modify a target.

The target may be a target nucleic acid, gene, chromosome or protein.

For example, the guide nucleic acid-editor protein complex may be used to ultimately regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein of interest, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a new protein.

Here, the guide nucleic acid-editor protein complex may act at a DNA, RNA, gene or chromosomal level.

For example, the guide nucleic acid-editor protein complex may regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein encoded by target DNA, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a modified protein through manipulation or modification of the target DNA.

In another example, the guide nucleic acid-editor protein complex may regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein encoded by target DNA, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a modified protein through manipulation or modification of target RNA.

In one example, the guide nucleic acid-editor protein complex may regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein encoded by target DNA, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a modified protein through manipulation or modification of a target gene.

In another example, the guide nucleic acid-editor protein complex may regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein encoded by target DNA, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a modified protein through manipulation or modification of a target chromosome.

The guide nucleic acid-editor protein complex may act at gene transcription and translation stages.

In one example, the guide nucleic acid-editor protein complex may promote or suppress the transcription of a target gene, thereby regulating (e.g., inhibiting, suppressing, reducing, increasing or promoting) the expression of a protein encoded by the target gene.

In another example, the guide nucleic acid-editor protein complex may promote or suppress the translation of a target gene, thereby regulating (e.g., inhibiting, suppressing, reducing, increasing or promoting) the expression of a protein encoded by the target gene.

The guide nucleic acid-editor protein complex may act at a protein level.

In one example, the guide nucleic acid-editor protein complex may manipulate or modify a target protein, thereby removing the target protein or regulating (e.g., inhibiting, suppressing, reducing, increasing or promoting) protein activity.

In one exemplary embodiment, the present invention provides a guide nucleic acid-editor protein complex used to manipulate a neovascularization-associated factor, for example, a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and/or an ANGPTL4 gene. Preferably, a gRNA-CRISPR enzyme complex is provided.

Particularly, the present invention may provide gRNA including a guide domain capable of forming a complementary bond with a target sequence from a gene, for example, isolated or non-natural gRNA and DNA encoding the same. The gRNA and the DNA sequence encoding the same may be designed to be able to complementarily bind to a target sequence listed in Table 1, Table 2, Table 3, Table 4 and Table 5.

In addition, a target region of the gRNA is designed to provide a third gene, which has a nucleic acid modification, for example, double or single strand breaks; or a specific function at a target site in a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and/or an ANGPTL4 gene.

In addition, when two or more gRNAs are used to induce two or more cleaving events in a target gene, for example, a double or single strand break, the two or more cleaving events may occur due to the same or different Cas9 proteins.

The gRNA may target, for example, two or more of the VEGFA gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and/or the ANGPTL4 gene, or two or more regions in each of the VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, and may independently induce the cleavage of a double strand and/or a single strand of the VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, or may induce the insertion of one foreign nucleotide into a cleavage site of the VEGFA gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and/or the ANGPTL4 gene.

In addition, in another exemplary embodiment of the present invention, a nucleic acid constituting the guide nucleic acid-editor protein complex may include: (a) a sequence encoding a guide nucleic acid including a guide domain, which is complementary to a target sequence of the VEGFA gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and/or the ANGPTL4 gene as described herein; and (b) a sequence encoding an editor protein.

Here, there may be two or more of the (a) according to a target region, and the (b) may employ the same or two or more editor proteins.

In an embodiment, the nucleic acid may be designed to target an enzymatically inactive editor protein or a fusion protein (e.g., a transcription repressor domain fusion) thereof to place it sufficiently adjacent to a knockdown target site in order to reduce, decrease or inhibit expression of the VEGFA gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and/or the ANGPTL4 gene.

Besides, it should be obvious that the above-described structure, function, and all applications of the guide nucleic acid-editor protein complex will be utilized in manipulation of the VEGFA gene, the HIF1A gene, the ANGPT2 gene, the EPAS1 gene, and/or the ANGPTL4 gene.

Use of Guide Nucleic Acid-Editor Protein Complex

In an embodiment for the use of the guide nucleic acid-editor protein complex of the present invention, the manipulation or modification of target DNA, RNA, genes or chromosomes using the gRNA-CRISPR enzyme complex will be described below.

Gene Manipulation

A target gene or nucleic acid may be manipulated or corrected using the above-described gRNA-CRISPR enzyme complex, that is, the CRISPR complex. Here, the manipulation or correction of the target gene or nucleic acid includes all of the stages of i) cleaving or damaging the target gene or nucleic acid and ii) repairing the damaged target gene or nucleic acid.

i) Cleavage or Damage of Target Gene or Nucleic Acid

i) The cleavage or damage of the target gene or nucleic acid may be cleavage or damage of the target gene or nucleic acid using the CRISPR complex, and particularly, cleavage or damage of a target sequence in the target gene or nucleic acid.

In one example, the cleavage or damage of the target gene or nucleic acid using the CRISPR complex may be complete cleavage or damage to the double strand of a target sequence.

In one exemplary embodiment, when wild-type SpCas9 is used, the double strand of a target sequence forming a complementary bond with gRNA may be completely cleaved.

In another exemplary embodiment, when SpCas9 nickase (D10A) and SpCas9 nickase (H840A) are used, a complementary single strand of a target sequence forming a complementary bond with gRNA may be cleaved by the SpCas9 nickase (D10A), and a non-complementary single strand of the target sequence forming a complementary bond with gRNA may be cleaved by the SpCas9 nickase (H840A), and the cleavages may take place sequentially or simultaneously.

In still another exemplary embodiment, when SpCas9 nickase (D10A) and SpCas9 nickase (H840A), and two gRNAs having different target sequences are used, a complementary single strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the SpCas9 nickase (D10A), a non-complementary single strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (H840A), and the cleavages may take place sequentially or simultaneously.

In another example, the cleavage or damage of a target gene or nucleic acid using the CRISPR complex may be cleavage or damage to only the single strand of a target sequence. Here, the single strand may be a complementary single strand of a target sequence forming a complementary bond with gRNA, or a non-complementary single strand of the target sequence forming a complementary bond with gRNA.

In one exemplary embodiment, when SpCas9 nickase (D10A) is used, a complementary single strand of a target sequence forming a complementary bond with gRNA may be cleaved by the SpCas9 nickase (D10A), but a non-complementary single strand of the target sequence forming a complementary bond with gRNA may not be cleaved.

In another exemplary embodiment, when SpCas9 nickase (H840A) is used, a complementary single strand of a target sequence forming a complementary bond with gRNA may be cleaved by the SpCas9 nickase (H840A), but a non-complementary single strand of the target sequence forming a complementary bond with gRNA may not be cleaved.

In yet another example, the cleavage or damage of a target gene or nucleic acid using the CRISPR complex may be partial removal of a nucleic acid fragment.

In one exemplary embodiment, when two gRNAs having different target sequences and wild-type SpCas9 are used, a double strand of a target sequence forming a complementary bond with the first gRNA may be cleaved, and a double strand of a target sequence forming a complementary bond with the second gRNA may be cleaved, resulting in the removal of nucleic acid fragments by the first and second gRNAs and SpCas9.

In another exemplary embodiment, when two gRNAs having different target sequences, wild-type SpCas9, SpCas9 nickase (D10A) and SpCas9 nickase (H840A) are used, a double strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the wild-type SpCas9, a complementary single strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (D10A), and a non-complementary single strand nay be cleaved by the SpCas9 nickase (H840A), resulting in the removal of nucleic acid fragments by the first and second gRNAs, the wild-type SpCas9, the SpCas9 nickase (D10A) and the SpCas9 nickase (H840A).

In still another exemplary embodiment, when two gRNAs having different target sequences, SpCas9 nickase (D10A) and SpCas9 nickase (H840A) are used, a complementary single strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the SpCas9 nickase (D10A), a non-complementary single strand may be cleaved by the SpCas9 nickase (H840A), a complementary double strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (D10A), and a non-complementary single strand may be cleaved by the SpCas9 nickase (H840A), resulting in the removal of nucleic acid fragments by the first and second gRNAs, the SpCas9 nickase (D10A) and the SpCas9 nickase (H840A).

In yet another exemplary embodiment, when three gRNAs having different target sequences, wild-type SpCas9, SpCas9 nickase (D10A) and SpCas9 nickase (H840A) are used, a double strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the wild-type SpCas9, a complementary single strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (D10A), and a non-complementary single strand of a target sequence forming a complementary bond with the third gRNA may be cleaved by the SpCas9 nickase (H840A), resulting in the removal of nucleic acid fragments by the first gRNA, the second gRNA, the third gRNA, the wild-type SpCas9, the SpCas9 nickase (D10A) and the SpCas9 nickase (H840A).

In yet another exemplary embodiment, when four gRNAs having different target sequences, SpCas9 nickase (D10A) and SpCas9 nickase (H840A) are used, a complementary single strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the SpCas9 nickase (D10A), a non-complementary single strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (H840A), a complementary single strand of a target sequence forming a complementary bond with the third gRNA may be cleaved by the SpCas9 nickase (D10A), and a non-complementary single strand of a target sequence forming a complementary bond with fourth gRNA may be cleaved by the SpCas9 nickase (H840A), resulting in the removal of nucleic acid fragments by the first gRNA, the second gRNA, the third gRNA, the fourth gRNA, the SpCas9 nickase (D10A) and the SpCas9 nickase (H840A).

ii) Repair or Restoration of Damaged Target Gene or Nucleic Acid

The target gene or nucleic acid cleaved or damaged by the CRISPR complex may be repaired or restored through NHEJ and homology-directed repairing (HDR).

Non-Homologous End Joining (NHEJ)

NHEJ is a method of restoration or repairing double strand breaks in DNA by joining both ends of a cleaved double or single strand together, and generally, when two compatible ends formed by breaking of the double strand (for example, cleavage) are frequently in contact with each other to completely join the two ends, the broken double strand is recovered. The NHEJ is a restoration method that is able to be used in the entire cell cycle, and usually occurs when there is no homologous genome to be used as a template in cells, like the G1 phase.

In the repair process of the damaged gene or nucleic acid using NHEJ, some insertions and/or deletions (indels) in the nucleic acid sequence occur in the NHEJ-repaired region, such insertions and/or deletions cause the leading frame to be shifted, resulting in frame-shifted transcriptome mRNA. As a result, innate functions are lost because of nonsense-mediated decay or the failure to synthesize normal proteins. In addition, while the leading frame is maintained, mutations in which insertion or deletion of a considerable amount of sequence may be caused to destroy the functionality of the proteins. The mutation is locus-dependent because mutations in a significant functional domain is probably less tolerated than mutations in a non-significant region of a protein.

While it is impossible to expect indel mutations produced by NHEJ in a natural state, a specific indel sequence is preferred in a given broken region, and can come from a small region of micro homology. Conventionally, the deletion length ranges from 1 bp to 50 bp, insertions tend to be shorter, and frequently include a short repeat sequence directly surrounding a broken region.

In addition, the NHEJ is a process causing a mutation, and when it is not necessary to produce a specific final sequence, may be used to delete a motif of the small sequence.

A specific knockout of a gene targeted by the CRISPR complex may be performed using such NHEJ. A double strand or two single strands of a target gene or nucleic acid may be cleaved using the CRISPR enzyme such as Cas9 or Cpf1, and the broken double strand or two single strands of the target gene or nucleic acid may have indels through the NHEJ, thereby inducing specific knockout of the target gene or nucleic acid. Here, the site of a target gene or nucleic acid cleaved by the CRISPR enzyme may be a non-coding or coding region, and in addition, the site of the target gene or nucleic acid restored by NHEJ may be a non-coding or coding region.

Homology Directed Repairing (HDR)

HDR is a correction method without an error, which uses a homologous sequence as a template to repair or restoration a damaged gene or nucleic acid, and generally, to repair or restoration broken DNA, that is, to restore innate information of cells, the broken DNA is repaired using information of a complementary base sequence which is not modified or information of a sister chromatid. The most common type of HDR is homologous recombination (HR). HDR is a repair or restoration method usually occurring in the S or G2/M phase of actively dividing cells.

To repair or restore damaged DNA using HDR, rather than using a complementary base sequence or sister chromatin of the cells, a DNA template artificially synthesized using information of a complementary base sequence or homologous base sequence, that is, a nucleic acid template including a complementary base sequence or homologous base sequence may be provided to the cells, thereby repairing the broken DNA. Here, when a nucleic acid sequence or nucleic acid fragment is further added to the nucleic acid template to repair the broken DNA, the nucleic acid sequence or nucleic acid fragment further added to the broken DNA may be subjected to knockin. The further added nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment for correcting the target gene or nucleic acid modified by a mutation to a normal gene or nucleic acid, or a gene or nucleic acid to be expressed in cells, but the present invention is not limited thereto.

In one example, a double or single strand of a target gene or nucleic acid may be cleaved using the CRISPR complex, a nucleic acid template including a base sequence complementary to a base sequence adjacent to the cleavage site may be provided to cells, and the cleaved base sequence of the target gene or nucleic acid may be repaired or restored through HDR.

Here, the nucleic acid template including the complementary base sequence may have broken DNA, that is, a cleaved double or single strand of a complementary base sequence, and further include a nucleic acid sequence or nucleic acid fragment to be inserted into the broken DNA. An additional nucleic acid sequence or nucleic acid fragment may be inserted into a cleaved site of the broken DNA, that is, the target gene or nucleic acid using the nucleic acid template including a nucleic acid sequence or nucleic acid fragment to be inserted into the complementary base sequence. Here, the nucleic acid sequence or nucleic acid fragment to be inserted and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment for correcting a target gene or nucleic acid modified by a mutation to a normal gene or nucleic acid or a gene or nucleic acid to be expressed in cells. The complementary base sequence may be a base sequence having complementary bonds with broken DNA, that is, right and left base sequences of the cleaved double or single strand of the target gene or nucleic acid. Alternatively, the complementary base sequence may be a base sequence having complementary bonds with broken DNA, that is, 3′ and 5′ ends of the cleaved double or single strand of the target gene or nucleic acid. The complementary base sequence may be a 15 to 3000-base sequence, a length or size of the complementary base sequence may be suitably designed according to a size of the nucleic acid template or the target gene. Here, as the nucleic acid template, a double- or single-stranded nucleic acid may be used, or it may be linear or circular, but the present invention is not limited thereto.

In another example, a double- or single-stranded target gene or nucleic acid is cleaved using the CRISPR complex, a nucleic acid template including a homologous base sequence with a base sequence adjacent to a cleavage site is provided to cells, and the cleaved base sequence of the target gene or nucleic acid may be repaired or restored by HDR.

Here, the nucleic acid template including the homologous base sequence may be broken DNA, that is, a cleaved double- or single-stranded homologous base sequence, and further include a nucleic acid sequence or nucleic acid fragment to be inserted into the broken DNA. An additional nucleic acid sequence or nucleic acid fragment may be inserted into broken DNA, that is, a cleaved site of a target gene or nucleic acid using the nucleic acid template including a homologous base sequence and a nucleic acid sequence or nucleic acid fragment to be inserted. Here, the nucleic acid sequence or nucleic acid fragment to be inserted and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment for correcting a target gene or nucleic acid modified by a mutation to a normal gene or nucleic acid or a gene or nucleic acid to be expressed in cells. The homologous base sequence may be broken DNA, that is, a base sequence having homology with cleaved double-stranded base sequence or right and left single-stranded base sequences of a target gene or nucleic acid. Alternatively, the complementary base sequence may be a base sequence having homology with broken DNA, that is, the 3′ and 5′ ends of a cleaved double or single strand of a target gene or nucleic acid. The homologous base sequence may be a 15 to 3000-base sequence, and a length or size of the homologous base sequence may be suitably designed according to a size of the nucleic acid template or a target gene or nucleic acid. Here, as the nucleic acid template, a double- or single-stranded nucleic acid may be used and may be linear or circular, but the present invention is not limited thereto.

Other than the NHEJ and HDR, there are methods of repairing or restoring broken DNA.

Single-Strand Annealing (SSA)

SSA is a method of repairing double strand breaks between two repeat sequences present in a target nucleic acid, and generally uses a repeat sequence of more than 30 bases. The repeat sequence is cleaved (to have sticky ends) to have a single strand with respect to a double strand of the target nucleic acid at each of the broken ends, and after the cleavage, a single-strand overhang containing the repeat sequence is coated with an RPA protein such that it is prevented from inappropriately annealing the repeat sequences to each other. RAD52 binds to each repeat sequence on the overhang, and a sequence capable of annealing a complementary repeat sequence is arranged. After annealing, a single-stranded flap of the overhang is cleaved, and synthesis of new DNA fills a certain gap to restore a DNA double strand. As a result of this repair, a DNA sequence between two repeats is deleted, and a deletion length may be dependent on various factors including the locations of the two repeats used herein, and a path or degree of the progress of cleavage.

SSA, similar to HDR, utilizes a complementary sequence, that is, a complementary repeat sequence, and in contrast, does not requires a nucleic acid template for modifying or correcting a target nucleic acid sequence.

Single-Strand Break Repair (SSBA)

Single strand breaks in a genome are repaired through a separate mechanism, SSBR, from the above-described repair mechanisms. In the case of single-strand DNA breaks, PARP1 and/or PARP2 recognizes the breaks and recruits a repair mechanism. PARP1 binding and activity with respect to the DNA breaks are temporary, and SSBR is promoted by promoting the stability of an SSBR protein complex in the damaged regions. The most important protein in the SSBR complex is XRCC1, which interacts with a protein promoting 3′ and 5′ end processing of DNA to stabilize the DNA. End processing is generally involved in repairing the damaged 3′ end to a hydroxylated state, and/or the damaged 5′ end to a phosphatic moiety, and after the ends are processed, DNA gap filling takes place. There are two methods for the DNA gap filling, that is, short patch repair and long patch repair, and the short patch repair involves insertion of a single base. After DNA gap filling, a DNA ligase promotes end joining.

Mismatch Repair (MMR)

MMR works on mismatched DNA bases. Each of an MSH2/6 or MSH2/3 complex has ATPase activity and thus plays an important role in recognizing a mismatch and initiating a repair, and the MSH2/6 primarily recognizes base-base mismatches and identifies one or two base mismatches, but the MSH2/3 primarily recognizes a larger mismatch.

Base Excision Repair (BER)

BER is a repair method which is active throughout the entire cell cycle, and used to remove a small non-helix-distorting base damaged region from the genome. In the damaged DNA, damaged bases are removed by cleaving an N-glycoside bond joining a base to the phosphate-deoxyribose backbone, and then the phosphodiester backbone is cleaved, thereby generating breaks in single-strand DNA. The broken single strand ends formed thereby were removed, a gap generated due to the removed single strand is filled with a new complementary base, and then an end of the newly-filled complementary base is ligated with the backbone by a DNA ligase, resulting in repair of the damaged DNA.

Nucleotide Excision Repair (NER)

NER is an excision mechanism important for removing large helix-distorting damage from DNA, and when the damage is recognized, a short single-strand DNA segment containing the damaged region is removed, resulting in a single strand gap of 22 to 30 bases. The generated gap is filled with a new complementary base, and an end of the newly filled complementary base is ligated with the backbone by a DNA ligase, resulting in the repair of the damaged DNA.

Gene Manipulation Effects

Manipulation or correction of a target gene or nucleic acid may largely lead to effects of knockout, knockdown, and knockin.

Knockout

The term “knockout” refers to inactivation of a target gene or nucleic acid, and the “inactivation of a target gene or nucleic acid” refers to a state in which transcription and/or translation of a target gene or nucleic acid does not occur. Transcription and translation of a gene causing a disease or a gene having an abnormal function may be inhibited through knockout, resulting in the prevention of protein expression.

For example, when a target gene or nucleic acid is edited or corrected using a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, the target gene or nucleic acid may be cleaved using the CRISPR complex. The damaged target gene or nucleic acid may be repaired through NHEJ using the CRISPR complex. The damaged target gene or nucleic acid may have indels due to NHEJ, and thereby, specific knockout for the target gene or nucleic acid may be induced.

Knockdown

The term “knockdown” refers to a decrease in transcription and/or translation of a target gene or nucleic acid or the expression of a target protein. The onset of a disease may be prevented or a disease may be treated by regulating the overexpression of a gene or protein through the knockdown.

For example, when a target gene or nucleic acid is edited or corrected using a gRNA-CRISPR inactive enzyme-transcription inhibitory activity domain complex, that is, a CRISPR inactive complex including a transcription inhibitory activity domain, the CRISPR inactive complex may specifically bind to the target gene or nucleic acid, transcription of the target gene or nucleic acid may be inhibited by the transcription inhibitory activity domain included in the CRISPR inactive complex, thereby inducing knockdown in which expression of the corresponding gene or nucleic acid is inhibited.

Knockin

The term “knockin” refers to insertion of a specific nucleic acid or gene into a target gene or nucleic acid, and here, the “specific nucleic acid” refers to a gene or nucleic acid of interest to be inserted or expressed. A mutant gene triggering a disease may be utilized in disease treatment by correction to normal or insertion of a normal gene to induce expression of the normal gene through the knockin.

In addition, the knockin may further need a donor.

For example, when a target gene or nucleic acid is edited or corrected using a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, the target gene or nucleic acid may be cleaved using the CRISPR complex. The target gene or nucleic acid damaged using the CRISPR complex may be repaired through HDR. Here, a specific nucleic acid may be inserted into the damaged gene or nucleic acid using a donor.

The term “donor” refers to a nucleic acid sequence that helps HDR-based repair of the damaged gene or nucleic acid, and here, the donor may include a specific nucleic acid.

The donor may be a double- or single-stranded nucleic acid.

The donor may be present in a linear or circular shape.

The donor may include a nucleic acid sequence having homology with a target gene or nucleic acid.

For example, the donor may include a nucleic acid sequence having homology with each of base sequences at a location into which a specific nucleic acid is to be inserted, for example, upstream (left) and downstream (right) of a damaged nucleic acid. Here, the specific nucleic acid to be inserted may be located between a nucleic acid sequence having homology with a base sequence downstream of the damaged nucleic acid and a nucleic acid sequence having homology with a base sequence upstream of the damaged nucleic acid. Here, the homologous nucleic acid sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homology or complete homology.

The donor may optionally include an additional nucleic acid sequence. Here, the additional nucleic acid sequence may serve to increase donor stability, knockin efficiency or HDR efficiency.

For example, the additional nucleic acid sequence may be an A, T-rich nucleic acid sequence, that is, an A-T rich domain. In addition, the additional nucleic acid sequence may be a scaffold/matrix attachment region (SMAR).

In one exemplary embodiment relating to a gene manipulation effect of the present invention, a manipulated target gene obtained using a gRNA-CRISPR enzyme complex, that is, a manipulated neovascularization-associated factor may have the following constitution.

In one exemplary embodiment, when the neovascularization-associated factor is a gene, the constitution of the artificially manipulated neovascularization-associated factor by the gRNA-CRISPR enzyme complex may include modification of one or more nucleic acids among a deletion or insertion of one or more nucleotides; a substitution with one or more nucleotides different from a wild-type gene; and an insertion of one or more foreign nucleotides in a continuous 1 bp to 50 bp, 1 bp to 40 bp or 1 bp to 30 bp, preferably, 3 bp to 25 bp region in the base sequence, which is located in a PAM sequence in a nucleic acid sequence constituting the neovascularization-associated factor or adjacent to a 5′ end and/or 3′ end thereof.

In addition, a chemical modification of one or more nucleotides may be included in the nucleic acid sequence constituting the neovascularization-associated factor.

Here, the “foreign nucleotide” is the concept including all exogeneous, for example, heterologous or artificially-synthesized nucleotides, other than nucleotides innately included in the neovascularization-associated factor. The foreign nucleotide also includes a nucleotide with a size of several hundred, thousand or tens of thousands of bp to express a protein having a specific function, as well as a small ologonucleotide with a size of 50 bp or less. Such a foreign nucleotide may be a donor.

The chemical modification may include methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation, for example, substitution of some functional groups contained in a nucleotide with any one of a hydrogen atom, a fluorine atom, an —O-alkyl group, an —O-acyl group, and an amino group, but the present invention is not limited thereto. In addition, to increase transferability of a nucleic acid molecule, the functional groups may also be substituted with any one of —Br, —Cl, —R, —R′OR, —SH, —SR, —N3 and —CN (R=alkyl, aryl, alkylene). In addition, the phosphate backbone of at least one nucleotide may be substituted with any one of an alkylphosphonate form, a phosphoroamidate form and a boranophosphate form. In addition, the chemical modification may be a substitution of at least one type of nucleotide contained in the nucleic acid molecule with any one of a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a morpholino, and a peptide nucleic acid (PNA), and the chemical modification may be bonding of the nucleic acid molecule with one or more selected from the group consisting of a lipid, a cell-penetrating peptide and a cell-target ligand.

To form a desired neovascularization regulating system, artificial modification using a gRNA-CRISPR enzyme complex may be applied to the nucleic acid constituting the neovascularization-associated factor.

A region including the nucleic acid modification of the neovascularization-associated factor may be a target region or target sequence.

Such a target sequence may be a target for the gRNA-CRISPR enzyme complex, and the target sequence may include or not include a PAM sequence recognized by the CRISPR enzyme. Such a target sequence may provide a critical standard in a gRNA designing stage to those of ordinary skill in the art.

Such nucleic acid modification includes the “cleavage” of a nucleic acid.

The term “cleavage” in a target region refers to breakage of a covalent backbone of polynucleotides. The cleavage includes enzymatic or chemical hydrolysis of a phosphodiester bond, but the present invention is not limited thereto, and also include various other methods. The cleavage is able to be performed on both of a single strand and a double strand, and the cleavage of a double strand may result from distinct single-strand cleavage. The double-strand cleavage may generate blunt ends or staggered ends.

When an inactivated CRISPR enzyme is used, it may induce a factor possessing a specific function to approach a certain region of the target region or neovascularization-associated factor without the cleavage process. Chemical modification of one or more nucleotides in the nucleic acid sequence of the neovascularization-associated factor may be included according to such a specific function.

In one example, various indels may occur due to target and non-target activities through the nucleic acid cleavage formed by the gRNA-CRISPR enzyme complex.

The term “indel” is the generic term for an insertion or deletion mutation occurring in-between some bases in a DNA base sequence. The indel may be introduced into a target sequence during repair by an HDR or NHEJ mechanism when the gRNA-CRISPR enzyme complex cleaves the nucleic acid (DNA or RNA) of the neovascularization-associated factor as described above.

The artificially manipulated neovascularization-associated factor of the present invention refers to modification of the nucleic acid sequence of an original gene by cleavage, indels, or insertion using a donor of such a nucleic acid, and contributes to a desired neovascularization regulating system, for example, exhibition of an effect of promoting or suppressing neovascularization.

For example, a specific protein may be expressed and its activity may be stimulated by the artificially manipulated neovascularization-associated factor.

A specific protein may be inactivated by the artificially manipulated neovascularization-associated factor.

In one example, a specific target region of each neovascularization-associated factor of the genome, for example, reverse regulatory genes such as a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and/or an ANGPTL4 gene may be cleaved, resulting in knockdown or knockout of the gene.

In another example, targeted knockdown may be mediated using an enzymatically inactive CRISPR enzyme fused to a transcription repressor domain or chromatin-modified protein to change transcription, for example, to block, negatively regulate or decrease the transcription of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene, and/or an ANGPTL4 gene.

The neovascularization may be regulated by the artificially manipulated neovascularization-associated factor.

A neovascularization-associated disease may be improved or treated by the artificially manipulated neovascularization-associated factor.

In one exemplary embodiment of the present invention, the artificially manipulated neovascularization-associated factor may provide various artificially manipulated neovascularization-associated factors according to the constitutional characteristic of the gRNA-CRISPR enzyme complex (e.g., included in a target region of the neovascularization-associated factor or different in the adjacent major PAM sequence).

Hereinafter, while representative examples of CRISPR enzymes and a neovascularization-regulatory gene have been illustrated, they are merely specific examples, and thus the present invention is not limited thereto.

For example, when the CRISPR enzyme is a SpCas9 protein, the PAM sequence is 5′-NGG-3′ (N is A, T, G, or C), and the cleaved base sequence region (target region) may be a continuous 1 bp to 25 bp, for example, 17 bp to 23 bp or 21 bp to 23 bp, region in the base sequence adjacent to the 5′ end and/or 3′ end of the 5′-NGG-3′ sequence in a target gene.

The present invention may provide an artificially manipulated neovascularization-associated factor, for example, an artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, which is prepared by

-   -   a) deletion of one or more nucleotides of a continuous 1 bp to         25 bp, for example, 17 bp to 23 bp, region in the base sequence         adjacent to the 5′ end and/or 3′ end of the 5′-NGG-3′ (N is A,         T, C or G) sequence,     -   b) substitution of one or more nucleotides of a continuous 1 bp         to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the 5′-NGG-3′         sequence with nucleotides different from those of the wild-type         gene,     -   c) insertion of one or more nucleotides into a continuous 1 bp         to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the 5′-NGG-3′         sequence, or     -   d) a combination of two or more selected from a) through c)     -   in the nucleic acid sequence of the         neovascularization-associated factor.

For example, when the CRISPR enzyme is a CjCas9 protein, the PAM sequence is 5′-NNNNRYAC-3′ (each N is independently A, T, C or G, R is A or G, and Y is C or T), and the cleaved base sequence region (target region) may be a continuous 1 bp to 25 bp, for example, 17 bp to 23 bp or 21 bp to 23 bp, region in the base sequence adjacent to the 5′ end and/or 3′ end of the 5′-NNNNRYAC-3′ sequence in a target gene.

The present invention may provide an artificially manipulated neovascularization-associated factor, for example, an artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, which is prepared by

-   -   a′) deletion of one or more nucleotides of a continuous 1 bp to         25 bp, for example, 17 bp to 23 bp, region in the base sequence         adjacent to the 5′ end and/or 3′ end of the 5′-NNNNRYAC-3′ (each         N is independently A, T, C or G, R is A or G, and Y is C or T),     -   b′) substitution of one or more nucleotides of a continuous 1 bp         to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the         5′-NNNNRYAC-3′ sequence with nucleotides different from those of         the wild-type gene,     -   c′) insertion of one or more nucleotides into a continuous 1 bp         to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the         5′-NNNNRYAC-3′ sequence, or     -   d′) a combination of two or more selected from a′) through c′)         in the nucleic acid sequence of the         neovascularization-associated factor.

For example, when the CRISPR enzyme is a StCas9 protein, the PAM sequence is 5′-NNAGAAW-3′ (each N is independently A, T, C or G, and W is A or T), and the cleaved base sequence region (target region) may be a continuous 1 bp to 25 bp, for example, 17 bp to 23 bp or 21 bp to 23 bp, region in the base sequence adjacent to the 5′ end and/or 3′ end of the 5′-NNAGAAW-3′ sequence in a target gene.

The present invention may provide an artificially manipulated neovascularization-associated factor, for example, an artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, which is prepared by

-   -   a″) deletion of one or more nucleotides of a continuous 1 bp to         25 bp, for example, 17 bp to 23 bp, region in the base sequence         adjacent to the 5′ end and/or 3′ end of the 5′-NNAGAAW-3′         sequence (each N is independently A, T, C or G, and W is A or         T),     -   b″) substitution of one or more nucleotides of a continuous 1 bp         to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the         5′-NNAGAAW-3′ sequence with nucleotides different from those of         the wild-type gene,     -   c″) insertion of one or more nucleotides into a continuous 1 bp         to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the         5′-NNAGAAW-3′ sequence, or     -   d″) a combination of two or more selected from a″) through c″)         in the nucleic acid sequence of the         neovascularization-associated factor.

For example, when the CRISPR enzyme is an NmCas9 protein, the PAM sequence is 5′-NNNNGATT-3′(each N is independently A, T, C or G), and the cleaved base sequence region (target region) may be a continuous 1 bp to 25 bp, for example, 17 bp to 23 bp or 21 bp to 23 bp, region in the base sequence adjacent to the 5′ end and/or 3′ end of the 5′-NNNNGATT-3′ sequence in a target gene.

The present invention may provide an artificially manipulated neovascularization-associated factor, for example, an artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, which is prepared by

-   -   a′″) deletion of one or more nucleotides of a continuous 1 bp to         25 bp, for example, 17 bp to 23 bp, region in the base sequence         adjacent to the 5′ end and/or the 3′ end of the 5′-NNNNGATT-3′         sequence (each N is independently A, T, C or G),     -   b′″) substitution of one or more nucleotides of a continuous 1         bp to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the         5′-NNNNGATT-3′ sequence with nucleotides different from those of         the wild-type gene,     -   c′″) insertion of one or more nucleotides into a continuous 1 bp         to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′-NNNNGATT-3′ sequence, or     -   d′″) a combination of two or more selected from a′″) through         c′″) in the nucleic acid sequence of the         neovascularization-associated factor.

For example, when the CRISPR enzyme is an SaCas9 protein, the PAM sequence is 5′-NNGRR(T)-3′ (each N is independently A, T, C or G, R is A or G, and (T) is a randomly addable sequence), and the cleaved base sequence region (target region) may be a continuous 1 bp to 25 bp, for example, 17 bp to 23 bp or 21 bp to 23 bp, region in the base sequence adjacent to the 5′ end and/or 3′ end of the 5′-NNGRR(T)-3′ sequence in a target gene.

The present invention may provide an artificially manipulated neovascularization-associated factor, for example, an artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, which is prepared by

a″″) deletion of one or more nucleotides of a continuous 1 bp to 25 bp, for example, 17 bp to 23 bp region, in the base sequence adjacent to the 5′ end and/or the 3′ end of the 5′-NNGRR(T)-3′ sequence (each N is independently A, T, C or G, R is A or G, and (T) is a randomly addable sequence),

-   -   b″″) substitution of one or more nucleotides of a continuous 1         bp to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the         5′-NNGRR(T)-3′ sequence with nucleotides different from those of         the wild-type gene,     -   c″″) insertion of one or more nucleotides into a continuous 1 bp         to 25 bp, for example, 17 bp to 23 bp, region in the base         sequence adjacent to the 5′-NNGRR(T)-3′ sequence, or     -   d″″) a combination of two or more selected from a″″) through         c″″) in the nucleic acid sequence of the         neovascularization-associated factor.

For example, when the CRISPR enzyme is a Cpf1 protein, the PAM sequence is 5′-TTN-3′ (N is A, T, C or G), and the cleaved base sequence region (target region) may be a continuous 10 bp to 30 bp, for example, 15 bp to 26 bp, 17 bp to 30 bp or 17 bp to 26 bp, region in the base sequence adjacent to the 5′ end or the 3′ end of the 5′-TTN-3′ sequence.

The Cpf1 protein may be derived from a microorganism such as Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium eligens, for example, Parcubacteria bacterium (GWC2011_GWC2_44_17), Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium eligens, but the present invention is not limited thereto.

The present invention may provide an artificially manipulated neovascularization-associated factor, for example, an artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene, which is prepared by

-   -   a″″″) deletion of one or more nucleotides of a continuous 10 bp         to 30 bp, for example, 15 bp to 26 bp, region in the base         sequence adjacent to the 5′ end and/or the 3′ end of the         5′-TTN-3′ sequence (N is A, T, C or G),     -   b″″″) substitution of one or more nucleotides of a continuous 10         bp to 30 bp, for example, 15 bp to 26 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the 5′-TTN-3′         sequence with nucleotides different from those of the wild-type         gene,     -   c″″″) insertion of one or more nucleotides of a continuous 10 bp         to 30 bp, for example, 15 bp to 26 bp, region in the base         sequence adjacent to the 5′ end and/or 3′ end of the 5′-TTN-3′         sequence, or     -   d″″″) a combination of two or more selected from a″″″) through         c″″″) in the nucleic acid sequence of the         neovascularization-associated factor.

In another exemplary embodiment, when the neovascularization-associated factor is a protein, the artificially manipulated protein includes all proteins involved in formation of new or modified blood vessels by a direct or indirect action of the gRNA-CRISPR enzyme complex.

For example, the artificially manipulated protein may be a protein expressed by a neovascularization-associated factor (gene) artificially manipulated by the gRNA-CRISPR enzyme complex or another protein increased or reduced by an influence by such protein activity, but the present invention is not limited thereto.

The artificially manipulated neovascularization-associated factor (protein) may have an amino acid composition and activity corresponding to the composition of the artificially manipulated neovascularization-associated factor (gene).

As an embodiment, an (i) artificially manipulated protein which is changed in expression characteristics may be provided.

For example, protein modification may have one or more characteristics:

-   -   a decrease or increase in expression level according to the         deletion or insertion of one or more nucleotides in a continuous         1 bp to 50 bp, 1 bp to 40 bp, 1 bp to 30 bp, and preferably 3 bp         to 25 bp region in the base sequence of the PAM sequence in the         nucleic acid sequence of the neovascularization-associated         factor or adjacent to the 5′ end and/or the 3′ end thereof;     -   a decrease or increase in expression level according to the         substitution with one or more nucleotides different from those         of a wild-type gene;     -   a decrease or increase in expression level, expression of a         fusion protein or independent expression of a specific protein         according to the insertion of one or more foreign nucleotides;         and     -   a decrease or increase in expression level of a third protein         influenced by expression characteristics of the above-described         proteins.

An (ii) artificially manipulated protein which is changed in structural characteristics may be provided.

For example, protein modification may have one or more characteristics:

-   -   a change in codons, amino acids and three-dimensional structure         according to the deletion or insertion of one or more         nucleotides in a continuous 1 bp to 50 bp, 1 bp to 40 bp, 1 bp         to 30 bp, and preferably 3 bp to 25 bp region in the base         sequence of the PAM sequence in the nucleic acid sequence of the         neovascularization-associated factor or adjacent to the 5′ end         and/or the 3′ end thereof;     -   a change in codons, amino acids, and three-dimensional structure         thereby according to the substitution with one or more         nucleotides different from a wild-type gene;     -   a change in codons, amino acids, and three-dimensional         structure, or a fusion structure with a specific protein or         independent structure from which a specific protein is separated         according to the insertion of one or more foreign nucleotides;         and     -   a change in codons, amino acids, and three-dimensional structure         of a third protein influenced by the above-described protein         changed in structural characteristic.

An (iii) artificially manipulated protein changed in functional characteristics may be provided.

For example, protein modification may have one or more characteristics:

-   -   the activation or inactivation of a specific function or         introduction of a new neovascularization function by protein         modification caused by a deletion or insertion of one or more         nucleotides in a continuous 1 bp to 50 bp, 1 bp to 40 bp, 1 bp         to 30 bp, and preferably 3 bp to 25 bp region in the base         sequence of the PAM sequence in the nucleic acid sequence of the         neovascularization-associated factor or adjacent to the 5′ end         and/or the 3′ end thereof;     -   the activation or inactivation of a specific function or         introduction of a new function by protein modification caused by         substitution with one or more nucleotides different from those         of a wild-type gene;     -   the activation or inactivation of a specific function or         introduction of a new function by protein modification caused by         insertion of one or more foreign nucleotides, particularly,         introduction of a third function to an existing function due to         fusion or independent expression of a specific protein; and     -   the change in the function of a third protein influenced by the         above-described protein changed in functional characteristics.

In addition, a protein artificially manipulated by the chemical modification of one or more nucleotides in the nucleic acid sequence constituting the neovascularization-associated factor may be included.

For example, one or more of the expression, structural and functional characteristics of a protein caused by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation and glycosylation may be changed.

For example, the third structure and function may be achieved by binding of a third protein into the nucleic acid sequence of the gene due to the chemical modification of nucleotides.

5. Other Additional Components

An additional component may be selectively added to increase the efficiency of a guide nucleic acid-editor protein complex or improve the repair efficiency of a damaged gene or nucleic acid.

The additional component may be selectively used to improve the efficiency of the guide nucleic acid-editor protein complex.

Activator

The additional component may be used as an activator to increase the cleavage efficiency of a target nucleic acid, gene or chromosome of the guide nucleic acid-editor protein complex.

The term “activator” refers to a nucleic acid serving to stabilize the bonding between the guide nucleic acid-editor protein complex and the target nucleic acid, gene or chromosome, or to allow the guide nucleic acid-editor protein complex to more easily approach the target nucleic acid, gene or chromosome.

The activator may be a double-stranded nucleic acid or single-stranded nucleic acid.

The activator may be linear or circular.

The activator may be divided into a “helper” that stabilizes the bonding between the guide nucleic acid-editor protein complex and the target nucleic acid, gene or chromosome, and an “escorter” that serves to allow the guide nucleic acid-editor protein complex to more easily approach the target nucleic acid, gene or chromosome.

The helper may increase the cleavage efficiency of the guide nucleic acid-editor protein complex with respect to the target nucleic acid, gene or chromosome.

For example, the helper includes a nucleic acid sequence having homology with the target nucleic acid, gene or chromosome. Therefore, when the guide nucleic acid-editor protein complex is bonded to the target nucleic acid, gene or chromosome, the homologous nucleic acid sequence included in the helper may form an additional complementary bond with the target nucleic acid, gene or chromosome to stabilize the bonding between the guide nucleic acid-editor protein complex and the target nucleic acid, gene or chromosome.

The escorter may increase the cleavage efficiency of the guide nucleic acid-editor protein complex with respect to the target nucleic acid, gene or chromosome.

For example, the escorter includes a nucleic acid sequence having homology with the target nucleic acid, gene or chromosome. Here, the homologous nucleic acid sequence included in the escorter may partly form a complementary bond with a guide nucleic acid of the guide nucleic acid-editor protein complex. Therefore, the escorter partly forming a complementary bond with the guide nucleic acid-editor protein complex may partly form a complementary bond with the target nucleic acid, gene or chromosome, and as a result, may allow the guide nucleic acid-editor protein complex to accurately approach the position of the target nucleic acid, gene or chromosome.

The homologous nucleic acid sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homology, or complete homology.

In addition, the additional component may be selectively used to improve the repair efficiency of the damaged gene or nucleic acid.

Assistor

The additional component may be used as an assistor to improve the repair efficiency of the damaged gene or nucleic acid.

The term “assistor” refers to a nucleic acid that serves to participate in a repair process or increase the repair efficiency of the damaged gene or nucleic acid, for example, the gene or nucleic acid cleaved by the guide nucleic acid-editor protein complex.

The assistor may be a double-stranded nucleic acid or single-stranded nucleic acid.

The assistor may be present in a linear or circular shape.

The assistor may be divided into an “NHEJ assistor” that participates in a repair process using NHEJ or improves repair efficiency and an “HDR assistor” that participates in a repair process using HDR or improves repair efficiency according to a repair method.

The NHEJ assistor may participate in a repair process or improve the repair efficiency of the damaged gene or nucleic acid using NHEJ.

For example, the NHEJ assistor may include a nucleic acid sequence having homology with a part of the damaged nucleic acid sequence. Here, the homologous nucleic acid sequence may include a nucleic acid sequence having homology with the nucleic acid sequence at one end (e.g., the 3′ end) of the damaged nucleic acid sequence, and include a nucleic acid sequence having homology with the nucleic acid sequence at the other end (e.g., the 5′ end) of the damaged nucleic acid sequence. In addition, a nucleic acid sequence having homology with each of the base sequences upstream and downstream of the damaged nucleic acid sequence may be included. The nucleic acid sequence having such homology may assist two parts of the damaged nucleic acid sequence to be placed in close proximity, thereby increasing the repair efficiency of the damaged nucleic acid by NHEJ.

The HDR assistor may participate in the repair process or improve repair efficiency of the damaged gene or nucleic acid using HDR.

For example, the HDR assistor may include a nucleic acid sequence having homology with a part of the damaged nucleic acid sequence. Here, the homologous nucleic acid sequence may include a nucleic acid sequence having homology with the nucleic acid sequence at one end (e.g., the 3′ end) of the damaged nucleic acid sequence, and a nucleic acid sequence having homology with the nucleic acid sequence at the other end (e.g., the 5′ end) of the damaged nucleic acid sequence. Alternatively, a nucleic acid sequence having homology with each of the base sequences upstream and downstream of the damaged nucleic acid sequence may be included. The nucleic acid sequence having such homology may serve as a template of the damaged nucleic acid sequence to increase the repair efficiency of the damaged nucleic acid by HDR.

In another example, the HDR assistor may include a nucleic acid sequence having homology with a part of the damaged nucleic acid sequence and a specific nucleic acid, for example, a nucleic acid or gene to be inserted. Here, the homologous nucleic acid sequence may include a nucleic acid sequence having homology with each of the base sequences upstream and downstream of the damaged nucleic acid sequence. The specific nucleic acid may be located between a nucleic acid sequence having homology with a base sequence downstream of the damaged nucleic acid and a nucleic acid sequence having homology with a base sequence upstream of the damaged nucleic acid. The nucleic acid sequence having such homology and specific nucleic acid may serve as a donor to insert a specific nucleic acid into the damaged nucleic acid, thereby increasing HDR efficiency for knockin.

The homologous nucleic acid sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homology or complete homology.

6. Subject

The term “subject” refers to an organism into which a guide nucleic acid, editor protein or guide nucleic acid-editor protein complex is introduced, an organism in which a guide nucleic acid, editor protein or guide nucleic acid-editor protein complex operates, or a specimen or sample obtained from the organism.

The subject may be an organism including a target nucleic acid, gene, chromosome or protein of the guide nucleic acid-editor protein complex.

The organism may be cells, tissue, a plant, an animal or a human.

The cells may be prokaryotic cells or eukaryotic cells.

The eukaryotic cells may be plant cells, animal cells or human cells, but the present invention is not limited thereto.

The tissue may be animal or human body tissue such as skin, liver, kidney, heart, lung, brain or muscle tissue.

The subject may be a specimen or sample including a target nucleic acid, gene, chromosome or protein of the guide nucleic acid-editor protein complex.

The specimen or sample may be obtained from an organism including a target nucleic acid, gene, chromosome or protein and may be saliva, blood, skin tissue, cancer cells or stem cells.

In the present invention, as a specific example, the subject may include a target gene or nucleic acid of the guide nucleic acid-editor protein complex.

Here, the target gene may be a neovascularization-associated factor, for example, a VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene.

The target gene may be a wild type, or a modified form in the wild-type.

In one exemplary embodiment of the present invention, the subject may include a gene or nucleic acid manipulated by the guide nucleic acid-editor protein complex.

Here, the manipulated gene may be a neovascularization-associated factor, for example, a VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene.

Here, the guide nucleic acid may target a neovascularization-associated factor, for example, a VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene.

The guide nucleic acid may be a nucleic acid sequence complementary to a target sequence of the VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene, and/or ANGPTL4 gene.

The guide nucleic acid may target one or more genes.

The guide nucleic acid may simultaneously target two or more genes. Here, the two or more genes may be homologous or heterologous genes.

The guide nucleic acid may target one or more target sequences.

The guide nucleic acid may be designed in various forms according to the number or locations of the target sequences.

In one exemplary embodiment of the present invention, the guide nucleic acid may be a nucleic acid sequence complementary to one or more target sequences of the sequences listed in Table 1, Table 2, Table 3, Table 4 and Table 5.

In a certain embodiment, for artificial manipulation of each gene, a guide nucleic acid sequence corresponding to any one of the target sequences of SEQ ID NOs: 1 to 79.

In a certain embodiment, for artificial manipulation of each gene, an editor protein that interacts with a guide nucleic acid sequence corresponding to, for example, forming a complex with any one of the target sequences of SEQ ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79, is provided.

In a certain embodiment, a nucleic acid modification product of each gene in which artificial manipulation occurs at a target sequence region of any one of SEQ ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79, and an expression product thereof are provided.

7. Delivery

The guide nucleic acid, editor protein or guide nucleic acid-editor protein complex may be delivered or introduced into a subject by various delivering methods and various forms.

The guide nucleic acid may be delivered or introduced into a subject in the form of DNA, RNA or a mixed form.

The editor protein may be delivered or introduced into a subject in the form of DNA, RNA, a DNA/RNA mixture, a peptide, a polypeptide, which encodes the editor protein, or a protein.

The guide nucleic acid-editor protein complex may be delivered or introduced into a target in the form of DNA, RNA or a mixture thereof, which encodes each component, that is, a guide nucleic acid or an editor protein.

The guide nucleic acid-editor protein complex may be delivered or introduced into a subject as a complex of a guide nucleic acid having a form of DNA, RNA or a mixture thereof and an editor protein having a form of a peptide, polypeptide or protein.

In addition, an additional component capable of increasing or inhibiting the efficiency of the guide nucleic acid-editor protein complex may be delivered or introduced into a subject by various delivering methods and in various forms.

The additional component may be delivered or introduced into a subject in the form of DNA, RNA, a DNA/RNA mixture, a peptide, a polypeptide or a protein.

i) Delivery in Form of DNA, RNA or Mixture Thereof

The form of DNA, RNA or a mixture thereof, which encodes the guide nucleic acid and/or editor protein may be delivered or introduced into a subject by a method known in the art.

Or, the form of DNA, RNA or a mixture thereof, which encodes the guide nucleic acid and/or editor protein may be delivered or introduced into a subject by a vector, a non-vector or a combination thereof.

The vector may be a viral or non-viral vector (e.g., a plasmid).

The non-vector may be naked DNA, a DNA complex or mRNA.

Vector-Based Introduction

The nucleic acid sequence encoding the guide nucleic acid and/or editor protein may be delivered or introduced into a subject by a vector.

The vector may include a nucleic acid sequence encoding a guide nucleic acid and/or editor protein.

For example, the vector may simultaneously include nucleic acid sequences, which encode the guide nucleic acid and the editor protein, respectively.

For example, the vector may include the nucleic acid sequence encoding the guide nucleic acid.

As an example, domains included in the guide nucleic acid may be contained all in one vector, or may be divided and then contained in different vectors.

For example, the vector may include the nucleic acid sequence encoding the editor protein.

In one example, in the case of the editor protein, the nucleic acid sequence encoding the editor protein may be contained in one vector, or may be divided and then contained in several vectors.

The vector may include one or more regulatory/control components.

Here, the regulatory/control components may include a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor and/or a 2A sequence.

The promoter may be a promoter recognized by RNA polymerase II.

The promoter may be a promoter recognized by RNA polymerase III.

The promoter may be an inducible promoter.

The promoter may be a subject-specific promoter.

The promoter may be a viral or non-viral promoter.

The promoter may use a suitable promoter according to a control region (that is, a nucleic acid sequence encoding a guide nucleic acid or editor protein).

For example, a promoter useful for the guide nucleic acid may be a H1, EF-1a, tRNA or U6 promoter. For example, a promoter useful for the editor protein may be a CMV, EF-1a, EFS, MSCV, PGK or CAG promoter.

The vector may be a viral vector or recombinant viral vector.

The virus may be a DNA virus or an RNA virus.

Here, the DNA virus may be a double-stranded DNA (dsDNA) virus or single-stranded DNA (ssDNA) virus.

Here, the RNA virus may be a single-stranded RNA (ssRNA) virus.

The virus may be a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia virus, a poxvirus or a herpes simplex virus, but the present invention is not limited thereto.

Generally, the virus may infect a host (e.g., cells), thereby introducing a nucleic acid encoding the genetic information of the virus into the host or inserting a nucleic acid encoding the genetic information into the host genome. The guide nucleic acid and/or editor protein may be introduced into a subject using a virus having such a characteristic. The guide nucleic acid and/or editor protein introduced using the virus may be temporarily expressed in the subject (e.g., cells). Alternatively, the guide nucleic acid and/or editor protein introduced using the virus may be continuously expressed in a subject (e.g., cells) for a long time (e.g., 1, 2 or 3 weeks, 1, 2, 3, 6 or 9 months, 1 or 2 years, or permanently).

The packaging capability of the virus may vary from at least 2 kb to 50 kb according to the type of virus. Depending on such a packaging capability, a viral vector including a guide nucleic acid or an editor protein or a viral vector including both of a guide nucleic acid and an editor protein may be designed. Alternatively, a viral vector including a guide nucleic acid, an editor protein and additional components may be designed.

In one example, a nucleic acid sequence encoding a guide nucleic acid and/or editor protein may be delivered or introduced by a recombinant lentivirus.

In another example, a nucleic acid sequence encoding a guide nucleic acid and/or editor protein may be delivered or introduced by a recombinant adenovirus.

In still another example, a nucleic acid sequence encoding a guide nucleic acid and/or editor protein may be delivered or introduced by recombinant AAV.

In yet another example, a nucleic acid sequence encoding a guide nucleic acid and/or editor protein may be delivered or introduced by a hybrid virus, for example, one or more hybrids of the virus listed herein.

Non-Vector-Based Introduction

A nucleic acid sequence encoding a guide nucleic acid and/or editor protein may be delivered or introduced into a subject using a non-vector.

The non-vector may include a nucleic acid sequence encoding a guide nucleic acid and/or editor protein.

The non-vector may be naked DNA, a DNA complex, mRNA, or a mixture thereof.

The non-vector may be delivered or introduced into a subject by electroporation, particle bombardment, sonoporation, magnetofection, transient cell compression or squeezing (e.g., described in the literature [Lee, et al, (2012) Nano Lett., 12, 6322-6327]), lipid-mediated transfection, a dendrimer, nanoparticles, calcium phosphate, silica, a silicate (Ormosil), or a combination thereof.

As an example, the delivery through electroporation may be performed by mixing cells and a nucleic acid sequence encoding a guide nucleic acid and/or editor protein in a cartridge, chamber or cuvette, and applying electrical stimuli with a predetermined duration and amplitude to the cells.

In another example, the non-vector may be delivered using nanoparticles. The nanoparticles may be inorganic nanoparticles (e.g., magnetic nanoparticles, silica, etc.) or organic nanoparticles (e.g., a polyethylene glycol (PEG)-coated lipid, etc.). The outer surface of the nanoparticles may be conjugated with a positively-charged polymer which is attachable (e.g., polyethyleneimine, polylysine, polyserine, etc.).

In a certain embodiment, the non-vector may be delivered using a lipid shell.

In a certain embodiment, the non-vector may be delivered using an exosome. The exosome is an endogenous nano-vesicle for transferring a protein and RNA, which can deliver RNA to the brain and another target organ.

In a certain embodiment, the non-vector may be delivered using a liposome. The liposome is a spherical vesicle structure which is composed of single or multiple lamellar lipid bilayers surrounding internal aqueous compartments and an external, lipophilic phospholipid bilayer which is relatively non-transparent. While the liposome may be made from several different types of lipids; phospholipids are most generally used to produce the liposome as a drug carrier.

Other additives may be included.

ii) Delivery in Form of Peptide, Polypeptide or Protein

An editor protein in the form of a peptide, polypeptide or protein may be delivered or introduced into a subject by a method known in the art

The peptide, polypeptide or protein form may be delivered or introduced into a subject by electroporation, microinjection, transient cell compression or squeezing (e.g., described in the literature [Lee, et al, (2012) Nano Lett., 12, 6322-6327]), lipid-mediated transfection, nanoparticles, a liposome, peptide-mediated delivery or a combination thereof.

The peptide, polypeptide or protein may be delivered with a nucleic acid sequence encoding a guide nucleic acid.

In one example, the transfer through electroporation may be performed by mixing cells into which the editor protein will be introduced with or without a guide nucleic acid in a cartridge, chamber or cuvette, and applying electrical stimuli with a predetermined duration and amplitude to the cells.

iii) Delivery in Form of Nucleic Acid-Protein Mixture

The guide nucleic acid and the editor protein may be delivered or introduced into a subject in the form of a guide nucleic acid-editor protein complex.

For example, the guide nucleic acid may be DNA, RNA or a mixture thereof. The editor protein may be a peptide, polypeptide or protein.

In one example, the guide nucleic acid and the editor protein may be delivered or introduced into a subject in the form of a guide nucleic acid-editor protein complex containing an RNA-type guide nucleic acid and a protein-type editor protein, that is, a ribonucleoprotein (RNP).

In the present invention, as an embodiment of a method for delivering the guide nucleic acid and/or editor protein into a subject, the delivery of gRNA, a CRISPR enzyme or a gRNA-CRISPR enzyme complex will be described below.

In an embodiment of the present invention, a nucleic acid sequence encoding the gRNA and/or CRISPR enzyme will be delivered or introduced into a subject using a vector.

The vector may include the nucleic acid sequence encoding the gRNA and/or CRISPR enzyme.

For example, the vector may simultaneously include the nucleic acid sequences encoding the gRNA and the CRISPR enzyme.

For example, the vector may include the nucleic acid sequence encoding the gRNA.

In one example, domains contained in the gRNA may be contained in one vector, or may be divided and then contained in different vectors.

For example, the vector may include the nucleic acid sequence encoding the CRISPR enzyme.

In one example, in the case of the CRISPR enzyme, the nucleic acid sequence encoding the CRISPR enzyme may be contained in one vector, or may be divided and then contained in several vectors.

The vector may include one or more regulatory/control components.

Here, the regulatory/control components may include a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor and/or a 2A sequence.

The promoter may be a promoter recognized by RNA polymerase II.

The promoter may be a promoter recognized by RNA polymerase III.

The promoter may be an inducible promoter.

The promoter may be a subject-specific promoter.

The promoter may be a viral or non-viral promoter.

The promoter may use a suitable promoter according to a control region (that is, a nucleic acid sequence encoding the gRNA and/or CRISPR enzyme).

For example, a promoter useful for the gRNA may be a H1, EF-1a, tRNA or U6 promoter. For example, a promoter useful for the CRISPR enzyme may be a CMV, EF-1a, EFS, MSCV, PGK or CAG promoter.

The vector may be a viral vector or recombinant viral vector.

The virus may be a DNA virus or an RNA virus.

Here, the DNA virus may be a double-stranded DNA (dsDNA) virus or single-stranded DNA (ssDNA) virus.

Here, the RNA virus may be a single-stranded RNA (ssRNA) virus.

The virus may be a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia virus, a poxvirus or a herpes simplex virus, but the present invention is not limited thereto.

Generally, the virus may infect a host (e.g., cells), thereby introducing a nucleic acid encoding the genetic information of the virus into the host or inserting a nucleic acid encoding the genetic information into the host genome. The gRNA and/or CRISPR enzyme may be introduced into a subject using a virus having such a characteristic. The gRNA and/or CRISPR enzyme introduced using the virus may be temporarily expressed in the subject (e.g., cells). Alternatively, the gRNA and/or CRISPR enzyme introduced using the virus may be continuously expressed in a subject (e.g., cells) for a long time (e.g., 1, 2 or 3 weeks, 1, 2, 3, 6 or 9 months, 1 or 2 years, or permanently).

The packaging capability of the virus may vary from at least 2 kb to 50 kb according to the type of virus. Depending on such a packaging capability, a viral vector only including gRNA or a CRISPR enzyme or a viral vector including both of gRNA and a CRISPR enzyme may be designed. Alternatively, a viral vector including gRNA, a CRISPR enzyme and additional components may be designed.

In one example, a nucleic acid sequence encoding gRNA and/or a CRISPR enzyme may be delivered or introduced by a recombinant lentivirus.

In another example, a nucleic acid sequence encoding gRNA and/or a CRISPR enzyme may be delivered or introduced by a recombinant adenovirus.

In still another example, a nucleic acid sequence encoding gRNA and/or a CRISPR enzyme may be delivered or introduced by recombinant AAV.

In yet another example, a nucleic acid sequence encoding gRNA and/or a CRISPR enzyme may be delivered or introduced by one or more hybrids of hybrid viruses, for example, the viruses described herein.

In one exemplary embodiment of the present invention, the gRNA-CRISPR enzyme complex may be delivered or introduced into a subject.

For example, the gRNA may be present in the form of DNA, RNA or a mixture thereof. The CRISPR enzyme may be present in the form of a peptide, polypeptide or protein.

In one example, the gRNA and CRISPR enzyme may be delivered or introduced into a subject in the form of a gRNA-CRISPR enzyme complex including RNA-type gRNA and a protein-type CRISPR, that is, a ribonucleoprotein (RNP).

The gRNA-CRISPR enzyme complex may be delivered or introduced into a subject by electroporation, microinjection, transient cell compression or squeezing (e.g., described in the literature [Lee, et al, (2012) Nano Lett., 12, 6322-6327]), lipid-mediated transfection, nanoparticles, a liposome, peptide-mediated delivery or a combination thereof.

8. Transformant

The term “transformant” refers to an organism into which a guide nucleic acid, editor protein or guide nucleic acid-editor protein complex is introduced, an organism in which a guide nucleic acid, editor protein or guide nucleic acid-editor protein complex is expressed, or a specimen or sample obtained from the organism.

The transformant may be an organism into which a guide nucleic acid, editor protein or guide nucleic acid-editor protein complex is introduced in the form of DNA, RNA or a mixture thereof.

For example, the transformant may be an organism into which a vector including a nucleic acid sequence encoding a guide nucleic acid and/or editor protein is introduced. Here, the vector may be a non-viral vector, viral vector or recombinant viral vector.

In another example, the transformant may be an organism into which a nucleic acid sequence encoding a guide nucleic acid and/or editor protein is introduced in a non-vector form. Here, the non-vector may be naked DNA, a DNA complex, mRNA or a mixture thereof.

The transformant may be an organism into which a guide nucleic acid, editor protein or guide nucleic acid-editor protein complex is introduced in the form of a peptide, polypeptide or protein.

The transformant may be an organism into which a guide nucleic acid, editor protein or guide nucleic acid-editor protein complex is introduced in the form of DNA, RNA, a peptide, a polypeptide, a protein or a mixture thereof.

For example, the transformant may be an organism into which a guide nucleic acid-editor protein complex including an RNA-type guide nucleic acid and a protein-type editor protein is introduced.

The transformant may be an organism including a target nucleic acid, gene, chromosome or protein of the guide nucleic acid-editor protein complex.

The organism may be cells, tissue, a plant, an animal or a human.

The cells may be prokaryotic cells or eukaryotic cells.

The eukaryotic cells may be plant cells, animal cells, or human cells, but the present invention is not limited thereto.

The tissue may be an animal or human body tissue such as skin, liver, kidney, heart, lung, brain, or muscle tissue.

The transformant may be an organism in(to)to which a guide nucleic acid, editor protein or guide nucleic acid-editor protein complex is introduced or expressed, or a specimen or sample obtained from the organism.

The specimen or sample may be saliva, blood, skin tissue, cancer cells or stem cells.

Use

One exemplary embodiment of the present invention relates to a use of treating a neovascularization-associated disease using a method of administering a composition for artificially manipulating a neovascularization-associated factor or an artificially manipulated neovascularization-associated factor to a subject.

Targets for the treatment may be mammals including primates such as a human or a monkey, rodents such as a mouse or a rat, etc.

Diseases to be Treated

In an embodiment, diseases to be treated may be neovascularization-associated diseases.

The term “neovascularization-associated diseases” refer to all states including excessive and/or abnormal neovascularization. The neovascularization-associated diseases refer to disorders characterized by vascularization which is not changed or regulated, except tumorigenesis or neoplastic transformation, that is, cancer. The neovascularization-associated diseases include an ocular neovascularization disease.

Neovascular diseases include neovascularization-dependent cancer, for example, solid tumors, hematomas such as leukemia and tumor metastasis; benign tumors such as hemangiomas, acoustic neuromas, neurofibroma, trachomas and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular neovascularization diseases such as diabetic retinopathy, retinopathy of prematurity, macular degenerations including dry age-related macular degeneration and wet age-related macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial neovascularization blindness; plaque neovascularization; telangiectasia; hemophiliac joint; angiofibromas; and wound granulation, but the present invention is not limited thereto.

In a certain embodiment, the neovascularization-associated diseases may be one or more diseases selected from the group consisting of rheumatoid arthritis, psoriasis, Osler-Webber Syndrome, myocardial neovascularization blindness, plaque neovascularization, telangiectasia, hemophiliac joint, angiofibromas, and wound granulation.

In an embodiment, the neovascularization-associated disease may be a disease including excessive and/or abnormal neovascularization.

In an embodiment, the neovascularization-associated disease may be neovascularization-dependent cancer.

Here, the neovascularization-dependent cancer includes solid tumors, hematologic tumors such as leukemia and tumor metastasis.

The neovascularization-dependent cancer may be, for example, solid tumors, hematologic tumors such as leukemia and tumor metastasis; benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachomas and pyogenic granulomas.

In a certain embodiment, the neovascularization-associated disease may be a benign tumor.

The benign tumor may include hemangiomas, acoustic neuromas, trachomas and pyogenic granulomas.

In a certain embodiment, the neovascularization-associated disease may be an ocular neovascularization disease.

The term “ocular neovascularization disease” refers to all ocular diseases including excessive and/or abnormal neovascularization. The ocular neovascularization disease includes a disorder characterized by vascularization that is not changed or regulated in the eyes.

As an example, the ocular neovascularization diseases may include: ischemic retinopathy, optic papillary neovascularization, iris neovascularization, retinal neovascularization, choroidal neovascularization, corneal neovascularization, vitreous neovascularization, glaucoma, panus, pterygiums, macular edemas, diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edemas, vascular retinopathy, retinal degeneration, uveitis, inflammatory diseases of the retina, and proliferative vitreoretinopathy.

In one exemplary embodiment, the ocular neovascularization disease may be diabetic retinopathy or macular degeneration.

Diabetic Retinopathy

Diabetic retinopathy is a diabetic complication occurring in approximately 40 to 45% of the patients diagnosed with any one of Type 1 diabetes or Type 2 diabetes.

Diabetic retinopathy usually affects both eyes, and generally progresses in four stages. The first stage, that is, mild nonproliferative retinopathy is characterized by ocular microaneurysms. Small scaled swelling occurs in a retinal capillary tube and a small vessel. In the second stage, that is, moderate nonproliferative retinopathy, a blood vessel provided to the retina starts to be blocked. In the third stage, that is, severe nonproliferative retinopathy, the occlusion leads to a decrease in blood supply to the retina, and the retina sends a neovascularization signal to the eye in order to provide blood supply to the retina. In the fourth stage, that is, proliferative retinopathy, which is the most advanced stage, angiogenesis occurs, but the new blood vessel is abnormal, weak, and grows on the surface of a vitreous gel contained in the retina and the eyes.

The diabetic retinopathy includes insulin-dependent diabetes, insulin-independent diabetes, retinal detachment, diabetic retinopathy, and vitreous hemorrhage.

Macular Degeneration

The macular degeneration refers to a disease in which visual impairment is caused by macular degeneration, and is also called age-related macular degeneration (AMD).

The AMD includes early, intermediate and advanced AMD, and also includes all of dry AMD, for example, geographic atrophy, and wet AMD which is also known as neovascular or exudative AMD.

Dry macular degeneration is a prevalent type accounting for approximately 90% of the AMDs when a lesion such as a druse (the state in which waste is accumulated in the macula) or retinal pigment epithelial atrophy is generated in the retina. Wet macular degeneration is characterized by production of choroidal neovascularization under the retina.

Dry macular degeneration includes macular degeneration caused by a missense mutation in an immunoregulatory complement factor H (CFH) gene.

In another exemplary embodiment, a use of a system for regulating an additional, third in vivo mechanism, accompanied with various functions of specific factors artificially modified in function (e.g., a gene known as a neovascularization-associated factor, etc.) may be provided.

For example, the specific factors artificially modified in function may be one or more genes of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene.

The third mechanism may be an in vivo mechanism in which the genes are involved, other than neovascularization.

Pharmaceutical Composition

One exemplary embodiment of the present invention relates to a composition to be used in treatment of a disease using an artificially manipulated neovascularization-associated factor.

The composition may include an artificially manipulated neovascularization-associated factor or a manipulation composition capable of artificially manipulating the neovascularization-associated factor. The composition may be referred to as a therapeutic composition or pharmaceutical composition.

In an exemplary embodiment, the composition may include an artificially manipulated neovascularization-associated factor, that is, a gene and/or protein.

In an exemplary embodiment, the composition may include a manipulation composition capable of artificially manipulating a neovascularization-associated factor.

The manipulation composition may include a guide nucleic acid-editor protein complex.

The manipulation composition may include a guide nucleic acid and/or editor protein.

The manipulation composition may include a nucleic acid encoding the guide nucleic acid and/or editor protein.

The manipulation composition may include a virus comprising a nucleic acid encoding the guide nucleic acid and/or editor protein.

In another exemplary embodiment, the composition may further include an additional element.

The additional element may include a suitable carrier for delivery into the body of a subject.

In one exemplary embodiment, the composition may include an expression product of a neovascularization-associated factor manipulated in a sufficient amount to suppress an angiovascular disorder.

The “sufficient amount to suppress an angiovascular disorder” refers to an effective amount necessary to treat or prevent an angiovascular disorder or a symptom thereof.

In one exemplary embodiment, the following therapeutic compositions will be provided:

-   -   a composition for treating an angiovascular disorder, which         includes a guide nucleic acid capable of forming a complementary         bond with each of one or more target sequences in nucleic acid         sequences of one or more genes selected from the group         consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an         EPAS1 gene and an ANGPTL4 gene, or a nucleic acid sequence         encoding the same, and     -   an editor protein or a nucleic acid sequence encoding the same;     -   a composition for treating an angiovascular disorder, which         includes a guide nucleic acid capable of forming a complementary         bond with each of the target sequences of SEQ ID NOs: 1 to 1522,         for example, SEQ ID NOs: 1 to 79, of nucleic acid sequences of         one or more genes selected from the group consisting of a VEGFA         gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an         ANGPTL4 gene, or a nucleic acid sequence encoding the same, and     -   an editor protein or a nucleic acid sequence encoding the same;         and     -   a composition for treating an angiovascular disorder, which         includes a complex formed of a guide nucleic acid capable of         forming a complementary bond with each of the target sequences         of SEQ ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79, of         nucleic acid sequences of one or more genes selected from the         group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene,         an EPAS1 gene and an ANGPTL4 gene or a nucleic acid sequence         encoding the same; and an editor protein.

Here, the guide nucleic acid or nucleic acid sequence encoding the same; and a nucleic acid sequence encoding the editor protein may be present in the form of one or more vectors. The guide nucleic acid or nucleic acid sequence encoding the same; and a nucleic acid sequence encoding the editor protein may be present in the form of homologous or heterologous vectors.

Treatment Method

In another exemplary embodiment of the present invention, a method for treating a disease in a patient, which includes producing the above-described composition and administering an effective amount of the composition to a patient requiring the same, is provided.

Gene Manipulating Treatment

A treatment method for regulating neovascularization by manipulating a gene of a living organism may be used. Such a treatment method may be achieved by directly injecting a composition for manipulating a gene to manipulate the gene of a living organism into the organism.

The composition for gene manipulation may include a guide nucleic acid-editor protein complex.

The composition for gene manipulation may be injected into a specific location of the body.

Here, the specific location of the body may be tissue in which neovascularization excessively and/or abnormally occurs, or a location close thereto. For example, the specific location of the body may be, for example, the eyeball.

Subjects for administration of the composition may be mammals including primates such as a human or a monkey, rodents such as a mouse or a rat, etc.

The composition may be administered by any convenient method such as injection, transfusion, implantation or transplantation. The composition may be administered subcutaneously, intradermally, intraocularly, intravitreally, intratumorally, intranodally, intramedullarily, intramuscularly, intravenously, intralymphatically, or intraperitoneally.

A dose (pharmaceutically effective amount to obtain a predetermined, desired effect) of the composition may be selected from all integers in the value ranges of 104-109 cells, for example, 105 to 106 cells/kg (body weight), per kg of the subject of administration, but the present invention is not limited thereto. The composition may be suitably prescribed in consideration of the age, health condition and body weight of the subject of administration, the types of treatments simultaneously received, if they were, frequency of the co-treatments, and characteristics of a desired effect.

In one aspect, the present invention provides a method for modifying a target polynucleotide in prokaryotic cells, which may be achieved in vivo, ex vivo, or in vitro.

In some embodiments, the method may include sampling cells or a cell population from a human or non-human animal, and modifying the cell or cells. Culturing may occur at any step ex vivo. The cell or cells may also be reintroduced into a non-human animal or plant. The reintroduced cells are most preferably stem cells.

In still another exemplary embodiment, the present invention may provide a method for artificially manipulating cells, which includes: introducing (a) a guide nucleic acid capable of forming a complementary bond with the target sequences of SEQ ID NOs: 1 to 1522, for example, SEQ ID NOs: 1 to 79, of nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene or a nucleic acid sequence encoding the same; and (b) an editor protein including one or more proteins selected from the group consisting of a Streptococcus pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived Cas9 protein, a Streptococcus aureus-derived Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a Cpf1 protein, or a nucleic acid sequence encoding the same to cells.

The guide nucleic acid and the editor protein may be present in one or more vectors in the form of a nucleic acid sequence, or in a complex of a combination of the guide nucleic acid and the editor protein.

The introduction step may be carried out in vivo or ex vivo.

A technique of the above-described “7. Delivery” section may be referenced before the introduction step.

For example, the introduction stage may be achieved by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles, and a protein translocation domain (PTD) fusion protein method.

For example, the viral vector may be one or more selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia virus, a poxvirus and a herpes simplex virus.

When a neovascularization-associated factor is artificially manipulated using the method and composition of some embodiments of the present invention, it is possible to regulate, for example, inhibit, suppress, stimulate and/or increase neovascularization, and therefore an effect of suppressing or improving excessive and/or abnormal neovascularization may be obtained.

Additional Uses

In a certain embodiment, the present invention may provide a kit for preparing a composition for treating AMD or diabetic retinopathy.

The kit may be prepared by a conventional preparation method known in the art.

The kit may further include a detectable label. The term “detectable label” refers to an atom or molecule for specifically detecting a molecule containing a label among the same type of molecules without a label. The detectable label may be attached to an antibody specifically binding to a protein or a fragment thereof, an interaction protein, a ligand, nanoparticles, or an aptamer. The detectable label may include a radionuclide, a fluorophore, and an enzyme.

In a certain embodiment, the present invention may provide a method for screening a material capable of regulating the expression level of one or more genes of an artificially manipulated VEGFA gene, HIF1A gene, ANGPT2 gene, EPAS1 gene and ANGPTL4 gene.

In a certain embodiment, the present invention may provide a method for providing information on the sequence of a target site which is able to be artificially manipulated in a subject by analyzing the sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene.

In addition, a method for constructing a library using the information provided by such a method.

Here, a known database may be used.

In specific embodiments, an animal or cells which can be used for research using the method of the present invention may be provided.

An animal or cells which includes chromosome editing in one or more nucleic acid sequences associated with a disease may be prepared using the above-described method. Such a nucleic acid sequence may be a reference sequence which may encode a disease-associated protein sequence or may be associated with a disease.

In one exemplary embodiment, an effect of mutation and occurrence and/or progression of a disease may be studied in an animal or cells using measurements conventionally used in disease research with the animal or cells prepared by the method of the present invention. Alternatively, a pharmaceutical effect of an active compound in a disease using such an animal or cells may be studied.

In another exemplary embodiment, the effect of the strategy of a possible gene therapy may be evaluated using the animal or cells prepared by the method of the present invention. That is, the development and/or progression of a corresponding disease may be suppressed or reduced by modifying a chromosome sequence encoding a disease-associated protein. Particularly, this method includes forming a modified protein by editing a chromosome sequence encoding a disease-associated protein, resulting in the achievement of a modified response of the animal or cells. Therefore, in some embodiments, a genetically-modified animal may be compared with an animal vulnerable to the development of a corresponding disease, thereby evaluating the effect of a gene therapy process.

Such uses may include a disease model, a pharmacological model, a developmental model, a cell function model, and a humanized model. For example, a neovascularization-associated disease model, a pharmacological model, a developmental model, a cell function model, and a humanized model may be included.

An artificially manipulated neovascularization-associated factor and a neovascularization system artificially modified in function thereby can be effectively used to treat a neovascularization-associated disease, for example, a neovascularization-associated ocular disease (eye disease). The efficiency of a neovascularization system may be improved by regulating various in vivo mechanisms in which various neovascularization-associated factors are involved.

Hereinafter, the present invention will be described in further detail with reference to examples.

The examples are merely provided to describe the present invention in further detail, and it might be obvious to those of ordinary skill in the art that the scope of the present invention is not limited to the following examples.

Experimental Methods

1. Design of sgRNA

CRISPR/Cas9 target regions of human VEGFA gene (NCBI Accession No. NM_001025366.2), HIF1A gene (NCBI Accession No. NM_001243084.1), ANGPT2 gene (NCBI Accession No. NM_001118887.1), EPAS1 gene (NCBI Accession No. NM_001430.4) and ANGPTL4 gene (NCBI Accession No. NM_001039667.2) were selected using CRISPR RGEN Tools (Institute for Basic Science, Korea). The target regions of the genes may be different according to the type of CRISPR enzyme. Target sequences of the genes for CjCas9 are summarized in Tables 1 to 5 and Tables 6 to 9 listed above, and target sequences of the genes for SpCas9 are summarized in Tables 10 to 14.

TABLE 6 Target sequences of VEGFA gene Gene No. Target sequence VEGFA 1 AAATCCCGGTATAAGTCCTGGA (SEQ ID NO: 80) VEGFA 2 GCACCAACGTACACGCTCCAGG (SEQ ID NO: 81) VEGFA 3 CATTAGACAGCAGCGGGCACCA (SEQ ID NO: 82) VEGFA 4 GGCATTAGACAGCAGCGGGCAC (SEQ ID NO: 83) VEGFA 5 GGCTCCAGGGCATTAGACAGCA (SEQ ID NO: 84) VEGFA 6 GCTCAGAGCGGAGAAAGCATTT (SEQ ID NO: 85) VEGFA 7 GGAACATTTACACGTCTGCGGA (SEQ ID NO: 86) VEGFA 8 GCAGACGTGTAAATGTTCCTGC (SEQ ID NO: 87) VEGFA 9 GAGTCTGTGTTTTTGCAGGAAC (SEQ ID NO: 88) VEGFA 10 GGCGAGGCAGCTTGAGTTAAAC (SEQ ID NO: 89)

TABLE 7 Target sequences of HIF1A gene Gene No. Target sequence HIF1A 1 ACTAAAGGACAAGTCACCACAG (SEQ ID NO: 90) HIF1A 2 TATCCACCTCTTTTGGCAAGCA (SEQ ID NO: 91) HIF1A 3 TGAAACTCAAGCAACTGTCATA (SEQ ID NO: 92) HIF1A 4 CTCACAACGTAATTCACACATA (SEQ ID NO: 93) HIF1A 5 TACTTACCTCACAACGTAATTC (SEQ ID NO: 94) HIF1A 6 AACTTACTTACCTCACAACGTA (SEQ ID NO: 95) HIF1A 7 TCTTGTTTTGACAGTGGTATTA (SEQ ID NO: 96) HIF1A 8 GGGAGAAAATCAAGTCGTGCTG (SEQ ID NO: 97) HIF1A 9 TATCTGAAGATTCAACCGGTTT (SEQ ID NO: 98) HIF1A 10 GCTATTCACCAAAGTTGAATCA (SEQ ID NO: 99) HIF1A 11 AACTTTGCTGGCCCCAGCCGCT (SEQ ID NO: 100) HIF1A 12 AAACTGATGACCAGCAACTTGA (SEQ ID NO: 101) HIF1A 13 GGGGAGCATTACATCATTATAT (SEQ ID NO: 102) HIF1A 14 AGCCACTTCGAAGTAGTGCTGA (SEQ ID NO: 103) HIF1A 15 CAACTTCTTGATTGAGTGCAGG (SEQ ID NO: 104) HIF1A 16 TTACCATGCCCCAGATTCAGGA (SEQ ID NO: 105) HIF1A 17 TCAGACACCTAGTCCTTCCGAT (SEQ ID NO: 106) HIF1A 18 ATTGGTAGAAAAACTTTTTGCT (SEQ ID NO: 107) HIF1A 19 AACTCATGTATTTGCTGTTTTA (SEQ ID NO: 108) HIF1A 20 AAGCCCTGAAAGCGCAAGTCCT (SEQ ID NO: 109) HIF1A 21 CAGTTACAGTATTCCAGCAGAC (SEQ ID NO: 110) HIF1A 22 AGGTTCTTGTATTTGAGTCTGC (SEQ ID NO: 111) HIF1A 23 ATGCAATCAATATTTTAATGTC (SEQ ID NO: 112) HIF1A 24 TGATTGCATCTCCATCTCCTAC (SEQ ID NO: 113) HIF1A 25 TAGTGCCACATCATCACCATAT (SEQ ID NO: 114) HIF1A 26 GAGTATCTCTATATGGTGATGA (SEQ ID NO: 115) HIF1A 27 ATACCTTTGACTCAAAGCGACA (SEQ ID NO: 116) HIF1A 28 TTCCTGAGGAAGAACTAAATCC (SEQ ID NO: 117) HIF1A 29 TCTGTTCACTAGATTTGCATCC (SEQ ID NO: 118) HIF1A 30 GAATGGAGCAAAAGACAATTAT (SEQ ID NO: 119) HIF1A 31 GTTATGATTGTGAAGTTAATGC (SEQ ID NO: 120)

TABLE 8 Target sequences of EPAS1 gene Gene No. Target sequence EPAS1 1 AACACCTCCGTCTCCTTGCTCC (SEQ ID NO: 121) EPAS1 2 TGGAGGCCTTGTCCAGATGGGA (SEQ ID NO: 122) EPAS1 3 TGCGACTGGCAATCAGCTTCCT (SEQ ID NO: 123) EPAS1 4 CGACTGGCAATCAGCTTCCTGC (SEQ ID NO: 124) EPAS1 5 GAAGCTGACCAGCAGATGGACA (SEQ ID NO: 125) EPAS1 6 GCAATGAAACCCTCCAAGGCTT (SEQ ID NO: 126) EPAS1 7 AAAACATCAGCAAGTTCATGGG (SEQ ID NO: 127) EPAS1 8 GCAAGTTCATGGGACTTACACA (SEQ ID NO: 128) EPAS1 9 GGTCGCAGGGATGAGTGAAGTC (SEQ ID NO: 129) EPAS1 10 GCGGGACTTCTTCATGAGGATG (SEQ ID NO: 130) EPAS1 11 GAAGTGCACGGTCACCAACAGA (SEQ ID NO: 131) EPAS1 12 ACAGTACGGCCTCTGTTGGTGA (SEQ ID NO: 132) EPAS1 13 TCCAGGTGGCTGACTTGAGGTT (SEQ ID NO: 133) EPAS1 14 TCCACGCCTGTCTCAGGTCTTG (SEQ ID NO: 134) EPAS1 15 CAGGACAGCAGGGGCTCCTTGT (SEQ ID NO: 135) EPAS1 16 CTCATCATCATGTGTGAACCAA (SEQ ID NO: 136) EPAS1 17 ATGTGGGATGGGTGCTGGATTG (SEQ ID NO: 137) EPAS1 18 GCCACTTACTACCTGACCCTTG (SEQ ID NO: 138) EPAS1 19 TGGCCACTTACTACCTGACCCT (SEQ ID NO: 139) EPAS1 20 ACCAAGGGTCAGGTAGTAAGTG (SEQ ID NO: 140) EPAS1 21 TAGCCCCCATGCTTTGCGAGCA (SEQ ID NO: 141) EPAS1 22 GATGACCGTCCCCTGGGTCTCC (SEQ ID NO: 142) EPAS1 23 CTCAGGACGTAGTTGACACACA (SEQ ID NO: 143) EPAS1 24 CATGCTTACCTCAGGACGTAGT (SEQ ID NO: 144) EPAS1 25 CACATGCTTACCTCAGGACGTA (SEQ ID NO: 145) EPAS1 26 CAGGGATTCAGTCTGGTCCATG (SEQ ID NO: 146) EPAS1 27 GGTGAATAGGAAGTTACTCTTC (SEQ ID NO: 147) EPAS1 28 ATGGGCCACGGAGTTGAGGAGC (SEQ ID NO: 148) EPAS1 29 CTAGCCCAATAGCCCTGAAGAC (SEQ ID NO: 149) EPAS1 30 AGTGATTGAGAAGCTCTTCGCC (SEQ ID NO: 150) EPAS1 31 GGACACAGAGGCCAAGGACCAA (SEQ ID NO: 151) EPAS1 32 CCTGATCTCCACAGCCATCTAC (SEQ ID NO: 152) EPAS1 33 CGGATTTCAATGAGCTGGACTT (SEQ ID NO: 153) EPAS1 34 TCAATGAGCTGGACTTGGAGAC (SEQ ID NO: 154) EPAS1 35 GCGGAGAACCCACAGTCCACCC (SEQ ID NO: 155) EPAS1 36 CCAGTGGCTGGAAGATGTTTGT (SEQ ID NO: 156) EPAS1 37 TTCCAGCCACTGGCCCCTGTAG (SEQ ID NO: 157) EPAS1 38 CTGGAGAGCAAGAAGACAGAGC (SEQ ID NO: 158) EPAS1 39 GAGAGAGGGGTGCTGGCCTGGC (SEQ ID NO: 159) EPAS1 40 CCTGCCACCGTGCTGTGGCCAG (SEQ ID NO: 160) EPAS1 41 TCTCTCTTCCATGGGGGGCAGA (SEQ ID NO: 161) EPAS1 42 CACAAAGTGGGCCGTCGGGGAT (SEQ ID NO: 162) EPAS1 43 GGAGAGGGCTACCATGGCCGGA (SEQ ID NO: 163) EPAS1 44 CTCAGGTCCTGGAAGGCTTGCT (SEQ ID NO: 164) EPAS1 45 CTCCCAGGGGGACCCACCTGGT (SEQ ID NO: 165) EPAS1 46 TGCCGGACAAGCCACTGAGCGC (SEQ ID NO: 166) EPAS1 47 TTCCCCCCACAGTGCTACGCCA (SEQ ID NO: 167) EPAS1 48 CTGTAGTCCTGGTACTGGGTGG (SEQ ID NO: 168) EPAS1 49 TGGGCTGACGACAGGCTGTAGT (SEQ ID NO: 169) EPAS1 50 TCCTTGCAGGAGCGTGGAGCTT (SEQ ID NO: 170)

TABLE 9 Target sequences of ANGPT2 gene Gene No. Target sequence ANGPT2 1 GACTGTGCTGAAGTATTCAAAT (SEQ ID NO: 171) ANGPT2 2 CTGTGCTGAAGTATTCAAATCA (SEQ ID NO: 172) ANGPT2 3 TGGTGTGTCCTGATTTGAATAC (SEQ ID NO: 173) ANGPT2 4 GCCATTCGTGGTGTGTCCTGAT (SEQ ID NO: 174) ANGPT2 5 ATCAGGACACACCACGAATGGC (SEQ ID NO: 175) ANGPT2 6 CTAATCAGCAACGCTATGTGCT (SEQ ID NO: 176) ANGPT2 7 AATCAGCAACGCTATGTGCTTA (SEQ ID NO: 177) ANGPT2 8 TTCCCAGTCTTTAAGGTGTATT (SEQ ID NO: 178) ANGPT2 9 TCTTCACTTGAGAGATAGAAAT (SEQ ID NO: 179) ANGPT2 10 CATCAGCCAACCAGGAAATGAT (SEQ ID NO: 180) ANGPT2 11 CTGTTAGCATTTGTGAACATTT (SEQ ID NO: 181) ANGPT2 12 TGTGGTCCTTCCAACTTGAACG (SEQ ID NO: 182) ANGPT2 13 CGGAATGTACTATCCACAGAGG (SEQ ID NO: 183) ANGPT2 14 TTATTTGTGTTCTGCCTCTGTG (SEQ ID NO: 184) ANGPT2 15 ACAAATAAGTTCAACGGCATTA (SEQ ID NO: 185) ANGPT2 16 AGCGAATAGCCTGAGCCTTTCC (SEQ ID NO: 186)

TABLE 10 Target sequences of VEGFA gene for SpCas9 Gene No. Target sequence VEGFA-Sp1 1  CAGCTGACCAGTCGCGCTGA (SEQ ID NO: 187) VEGFA-Sp2 2 GTGGTAGCTGGGGCTGGGGG (SEQ ID NO: 188) VEGFA-Sp3 3 GAGGTGGTAGCTGGGGCTGG (SEQ ID NO: 189) VEGFA-Sp4 4 GGAGGTGGTAGCTGGGGCTG (SEQ ID NO: 190) VEGFA-Sp5 5 AGGAGGTGGTAGCTGGGGCT (SEQ ID NO: 191) VEGFA-Sp6 6 GAGGAGGTGGTAGCTGGGGC (SEQ ID NO: 192) VEGFA-Sp7 7 CGGGGAGGAGGTGGTAGCTG (SEQ ID NO: 193) VEGFA-Sp8 8 CCCAGCTACCACCTCCTCCC (SEQ ID NO: 194) VEGFA-Sp9 9 CCGGGGAGGAGGTGGTAGCT (SEQ ID NO: 195) VEGFA-Sp10 10 GCCGGGGAGGAGGTGGTAGC (SEQ ID NO: 196) VEGFA-Sp11 11 GCTACCACCTCCTCCCCGGC (SEQ ID NO: 197) VEGFA-Sp12 12 ACCACCTCCTCCCCGGCCGG (SEQ ID NO: 198) VEGFA-Sp13 13 GCCGCCGGCCGGGGAGGAGG (SEQ ID NO: 199) VEGFA-Sp14 14 ACCTCCTCCCCGGCCGGCGG (SEQ ID NO: 200) VEGFA-Sp15 15 TCCGCCGCCGGCCGGGGAGG (SEQ ID NO: 201) VEGFA-Sp16 16 CTGTCCGCCGCCGGCCGGGG (SEQ ID NO: 202) VEGFA-Sp17 17 CCCCGGCCGGCGGCGGACAG (SEQ ID NO: 203) VEGFA-Sp18 18 CCACTGTCCGCCGCCGGCCG (SEQ ID NO: 204) VEGFA-Sp19 19 TCCACTGTCCGCCGCCGGCC (SEQ ID NO: 205) VEGFA-Sp20 20 GTCCACTGTCCGCCGCCGGC (SEQ ID NO: 206) VEGFA-Sp21 21 CCGGCGGCGGACAGTGGACG (SEQ ID NO: 207) VEGFA-Sp22 22 CCGCGTCCACTGTCCGCCGC (SEQ ID NO: 208) VEGFA-Sp23 23 GCGGCGGACAGTGGACGCGG (SEQ ID NO: 209) VEGFA-Sp24 24 GTGGACGCGGCGGCGAGCCG (SEQ ID NO: 210) VEGFA-Sp25 25 TGGACGCGGCGGCGAGCCGC (SEQ ID NO: 211) VEGFA-Sp26 26 CGCGGCGGCGAGCCGCGGGC (SEQ ID NO: 212) VEGFA-Sp27 27 GCGGCGGCGAGCCGCGGGCA (SEQ ID NO: 213) VEGFA-Sp28 28 CGGCGGCGAGCCGCGGGCAG (SEQ ID NO: 214) VEGFA-Sp29 29 GGCGAGCCGCGGGCAGGGGC (SEQ ID NO: 215) VEGFA-Sp30 30 CGGGCTCCGGCCCCTGCCCG (SEQ ID NO: 216) VEGFA-Sp31 31 CAGGGGCCGGAGCCCGCGCC (SEQ ID NO: 217) VEGFA-Sp32 32 GGGCCGGAGCCCGCGCCCGG (SEQ ID NO: 218) VEGFA-Sp33 33 CCGGAGCCCGCGCCCGGAGG (SEQ ID NO: 219) VEGFA-Sp34 34 CCGCCTCCGGGCGCGGGCTC (SEQ ID NO: 220) VEGFA-Sp35 35 CGGAGCCCGCGCCCGGAGGC (SEQ ID NO: 221) VEGFA-Sp36 36 GGAGCCCGCGCCCGGAGGCG (SEQ ID NO: 222) VEGFA-Sp37 37 GCCCGCGCCCGGAGGCGGGG (SEQ ID NO: 223) VEGFA-Sp38 38 TCCACCCCGCCTCCGGGCGC (SEQ ID NO: 224) VEGFA-Sp39 39 CTCCACCCCGCCTCCGGGCG (SEQ ID NO: 225) VEGFA-Sp40 40 CGCGCCCGGAGGCGGGGTGG (SEQ ID NO: 226) VEGFA-Sp41 41 GCGCCCGGAGGCGGGGTGGA (SEQ ID NO: 227) VEGFA-Sp42 42 CGCCCGGAGGCGGGGTGGAG (SEQ ID NO: 228) VEGFA-Sp43 43 GCCCGGAGGCGGGGTGGAGG (SEQ ID NO: 229) VEGFA-Sp44 44 ACCCCCTCCACCCCGCCTCC (SEQ ID NO: 230) VEGFA-Sp45 45 GACCCCCTCCACCCCGCCTC (SEQ ID NO: 331) VEGFA-Sp46 46 GGAGGCGGGGTGGAGGGGGT (SEQ ID NO: 232) VEGFA-Sp47 47 GAGGCGGGGTGGAGGGGGTC (SEQ ID NO: 233) VEGFA-Sp48 48 AGGCGGGGTGGAGGGGGTCG (SEQ ID NO: 234) VEGFA-Sp49 49 GTGGAGGGGGTCGGGGCTCG (SEQ ID NO: 235) VEGFA-Sp50 50 GAAACTTTTCGTCCAACTTC (SEQ ID NO: 236) VEGFA-Sp51 51 AAACTTTTCGTCCAACTTCT (SEQ ID NO: 237) VEGFA-Sp52 52 AGCGAGAACAGCCCAGAAGT (SEQ ID NO: 238) VEGFA-Sp53 53 CTTCTGGGCTGTTCTCGCTT (SEQ ID NO: 239) VEGFA-Sp54 54 CTGGGCTGTTCTCGCTTCGG (SEQ ID NO: 240) VEGFA-Sp55 55 TTCTCGCTTCGGAGGAGCCG (SEQ ID NO: 241) VEGFA-Sp56 56 CGGAGGAGCCGTGGTCCGCG (SEQ ID NO: 242) VEGFA-Sp57 57 GGAGGAGCCGTGGTCCGCGC (SEQ ID NO: 243) VEGFA-Sp58 58 GAGGAGCCGTGGTCCGCGCG (SEQ ID NO: 244) VEGFA-Sp59 59 AGGAGCCGTGGTCCGCGCGG (SEQ ID NO: 245) VEGFA-Sp60 60 GGCTTCCCCCGCGCGGACCA (SEQ ID NO: 246) VEGFA-Sp61 61 TCGGCTCGGCTTCCCCCGCG (SEQ ID NO: 247) VEGFA-Sp62 62 GCGGGGGAAGCCGAGCCGAG (SEQ ID NO: 248) VEGFA-Sp63 63 TCTCGCGGCTCCGCTCGGCT (SEQ ID NO: 249) VEGFA-Sp64 64 GCACTTCTCGCGGCTCCGCT (SEQ ID NO: 250) VEGFA-Sp65 65 AGCCGCGAGAAGTGCTAGCT (SEQ ID NO: 251) VEGFA-Sp66 66 GCCGCGAGAAGTGCTAGCTC (SEQ ID NO: 252) VEGFA-Sp67 67 GCCCGAGCTAGCACTTCTCG (SEQ ID NO: 253) VEGFA-Sp68 68 CGAGAAGTGCTAGCTCGGGC (SEQ ID NO: 254) VEGFA-Sp69 69 GAGAAGTGCTAGCTCGGGCC (SEQ ID NO: 255) VEGFA-Sp70 70 AAGTGCTAGCTCGGGCCGGG (SEQ ID NO: 256) VEGFA-Sp71 71 GGGCCGGGAGGAGCCGCAGC (SEQ ID NO: 257) VEGFA-Sp72 72 CCGGGAGGAGCCGCAGCCGG (SEQ ID NO: 258) VEGFA-Sp73 73 CCTCCGGCTGCGGCTCCTCC (SEQ ID NO: 259) VEGFA-Sp74 74 GGAGGAGCCGCAGCCGGAGG (SEQ ID NO: 260) VEGFA-Sp75 75 GAGGAGCCGCAGCCGGAGGA (SEQ ID NO: 261) VEGFA-Sp76 76 AGGAGCCGCAGCCGGAGGAG (SEQ ID NO: 262) VEGFA-Sp77 77 GGAGCCGCAGCCGGAGGAGG (SEQ ID NO: 263) VEGFA-Sp78 78 GCCGCAGCCGGAGGAGGGGG (SEQ ID NO: 264) VEGFA-Sp79 79 TCCTCCCCCTCCTCCGGCTG (SEQ ID NO: 265) VEGFA-Sp80 80 GCAGCCGGAGGAGGGGGAGG (SEQ ID NO: 266) VEGFA-Sp81 81 TCTTCCTCCTCCCCCTCCTC (SEQ ID NO: 267) VEGFA-Sp82 82 GAAGAGAAGGAAGAGGAGAG (SEQ ID NO: 268) VEGFA-Sp83 83 AAGAGAAGGAAGAGGAGAGG (SEQ ID NO: 269) VEGFA-Sp84 84 AAGAGGAGAGGGGGCCGCAG (SEQ ID NO: 270) VEGFA-Sp85 85 AGGGGGCCGCAGTGGCGACT (SEQ ID NO: 271) VEGFA-Sp86 86 CGAGCGCCGAGTCGCCACTG (SEQ ID NO: 272) VEGFA-Sp87 87 CGCAGTGGCGACTCGGCGCT (SEQ ID NO: 273) VEGFA-Sp88 88 GCGACTCGGCGCTCGGAAGC (SEQ ID NO: 274) VEGFA-Sp89 89 CGACTCGGCGCTCGGAAGCC (SEQ ID NO: 275) VEGFA-Sp90 90 GCGCTCGGAAGCCGGGCTCA (SEQ ID NO: 276) VEGFA-Sp91 91 TCGGAAGCCGGGCTCATGGA (SEQ ID NO: 277) VEGFA-Sp92 92 CGGAAGCCGGGCTCATGGAC (SEQ ID NO: 278) VEGFA-Sp93 93 GCCGGGCTCATGGACGGGTG (SEQ ID NO: 279) VEGFA-Sp94 94 GCCTCACCCGTCCATGAGCC (SEQ ID NO: 280) VEGFA-Sp95 95 GGGCTCATGGACGGGTGAGG (SEQ ID NO: 281) VEGFA-Sp96 96 CTCATGGACGGGTGAGGCGG (SEQ ID NO: 282) VEGFA-Sp97 97 TCCAGCCGCGCGCGCTCCCC (SEQ ID NO: 283) VEGFA-Sp98 98 GCCTGGGGAGCGCGCGCGGC (SEQ ID NO: 284) VEGFA-Sp99 99 CAGGGCCTGGGGAGCGCGCG (SEQ ID NO: 285) VEGFA-Sp100 100 CGCGCGCGCTCCCCAGGCCC (SEQ ID NO: 286) VEGFA-Sp101 101 GCGCTCCCCAGGCCCTGGCC (SEQ ID NO: 287) VEGFA-Sp102 102 CGCTCCCCAGGCCCTGGCCC (SEQ ID NO: 288) VEGFA-Sp103 103 GAGGCCCGGGCCAGGGCCTG (SEQ ID NO: 289) VEGFA-Sp104 104 CGAGGCCCGGGCCAGGGCCT (SEQ ID NO: 290) VEGFA-Sp105 105 CCAGGCCCTGGCCCGGGCCT (SEQ ID NO: 291) VEGFA-Sp106 106 CCGAGGCCCGGGCCAGGGCC (SEQ ID NO: 292) VEGFA-Sp107 107 CAGGCCCTGGCCCGGGCCTC (SEQ ID NO: 293) VEGFA-Sp108 108 CCCTGGCCCGGGCCTCGGGC (SEQ ID NO: 294) VEGFA-Sp109 109 CCGGCCCGAGGCCCGGGCCA (SEQ ID NO: 295) VEGFA-Sp110 110 CCTGGCCCGGGCCTCGGGCC (SEQ ID NO: 296) VEGFA-Sp111 111 CCCGGCCCGAGGCCCGGGCC (SEQ ID NO: 297) VEGFA-Sp112 112 CTGGCCCGGGCCTCGGGCCG (SEQ ID NO: 298) VEGFA-Sp113 113 GCCCGGGCCTCGGGCCGGGG (SEQ ID NO: 299) VEGFA-Sp114 114 TCCTCCCCGGCCCGAGGCCC (SEQ ID NO: 300) VEGFA-Sp115 115 TTCCTCCCCGGCCCGAGGCC (SEQ ID NO: 301) VEGFA-Sp116 116 TACTCTTCCTCCCCGGCCCG (SEQ ID NO: 302) VEGFA-Sp117 117 GGCGAGCTACTCTTCCTCCC (SEQ ID NO: 303) VEGFA-Sp118 118 GGAGGAAGAGTAGCTCGCCG (SEQ ID NO: 304) VEGFA-Sp119 119 AGTAGCTCGCCGAGGCGCCG (SEQ ID NO: 305) VEGFA-Sp120 120 CGCCGAGGCGCCGAGGAGAG (SEQ ID NO: 306) VEGFA-Sp121 121 GCCGAGGCGCCGAGGAGAGC (SEQ ID NO: 307) VEGFA-Sp122 122 GCCCGCTCTCCTCGGCGCCT (SEQ ID NO: 308) VEGFA-Sp123 123 GTGGGGCGGCCCGCTCTCCT (SEQ ID NO: 309) VEGFA-Sp124 124 GGCCGCCCCACAGCCCGAGC (SEQ ID NO: 310) VEGFA-Sp125 125 CTCCGGCTCGGGCTGTGGGG (SEQ ID NO: 311) VEGFA-Sp126 126 CCCCACAGCCCGAGCCGGAG (SEQ ID NO: 312) VEGFA-Sp127 127 CCTCTCCGGCTCGGGCTGTG (SEQ ID NO: 313) VEGFA-Sp128 128 CCCACAGCCCGAGCCGGAGA (SEQ ID NO: 314) VEGFA-Sp129 129 CCCTCTCCGGCTCGGGCTGT (SEQ ID NO: 315) VEGFA-Sp130 130 TCCCTCTCCGGCTCGGGCTG (SEQ ID NO: 316) VEGFA-Sp131 131 TCGCGCTCCCTCTCCGGCTC (SEQ ID NO: 317) VEGFA-Sp132 132 CTCGCGCTCCCTCTCCGGCT (SEQ ID NO: 318) VEGFA-Sp133 133 CGCGGCTCGCGCTCCCTCTC (SEQ ID NO: 319) VEGFA-Sp134 134 AGAGGGAGCGCGAGCCGCGC (SEQ ID NO: 320) VEGFA-Sp135 135 CGAGCCGCGCCGGCCCCGGT (SEQ ID NO: 321) VEGFA-Sp136 136 GAGCCGCGCCGGCCCCGGTC (SEQ ID NO: 322) VEGFA-Sp137 137 AGGCCCGACCGGGGCCGGCG (SEQ ID NO: 323) VEGFA-Sp138 138 TTCGGAGGCCCGACCGGGGC (SEQ ID NO: 324) VEGFA-Sp139 139 TGGTTTCGGAGGCCCGACCG (SEQ ID NO: 325) VEGFA-Sp140 140 ATGGTTTCGGAGGCCCGACC (SEQ ID NO: 326) VEGFA-Sp141 141 CATGGTTTCGGAGGCCCGAC (SEQ ID NO: 327) VEGFA-Sp142 142 CAGAAAGTTCATGGTTTCGG (SEQ ID NO: 328) VEGFA-Sp143 143 CAGCAGAAAGTTCATGGTTT (SEQ ID NO: 329) VEGFA-Sp144 144 CCATGAACTTTCTGCTGTCT (SEQ ID NO: 330) VEGFA-Sp145 145 CCAAGACAGCAGAAAGTTCA (SEQ ID NO: 331) VEGFA-Sp146 146 CATGAACTTTCTGCTGTCTT (SEQ ID NO: 332) VEGFA-Sp147 147 TTCTGCTGTCTTGGGTGCAT (SEQ ID NO: 333) VEGFA-Sp148 148 GGAGGTAGAGCAGCAAGGCA (SEQ ID NO: 334) VEGFA-Sp149 149 ATGGTGGAGGTAGAGCAGCA (SEQ ID NO: 335) VEGFA-Sp150 150 GCTCTACCTCCACCATGCCA (SEQ ID NO: 336) VEGFA-Sp151 151 CGCTTACCTTGGCATGGTGG (SEQ ID NO: 337) VEGFA-Sp152 152 GACCGCTTACCTTGGCATGG (SEQ ID NO: 338) VEGFA-Sp153 153 CACGACCGCTTACCTTGGCA (SEQ ID NO: 339) VEGFA-Sp154 154 TTTCTGTCCTCAGTGGTCCC (SEQ ID NO: 340) VEGFA-Sp155 155 GGTGCAGCCTGGGACCACTG (SEQ ID NO: 341) VEGFA-Sp156 156 GTGGTCCCAGGCTGCACCCA (SEQ ID NO: 342) VEGFA-Sp157 157 TTCTGCCATGGGTGCAGCCT (SEQ ID NO: 343) VEGFA-Sp158 158 CTTCTGCCATGGGTGCAGCC (SEQ ID NO: 344) VEGFA-Sp159 159 CAGGCTGCACCCATGGCAGA (SEQ ID NO: 345) VEGFA-Sp160 160 GCTGCACCCATGGCAGAAGG (SEQ ID NO: 346) VEGFA-Sp161 161 GCACCCATGGCAGAAGGAGG (SEQ ID NO: 347) VEGFA-Sp162 162 CACCCATGGCAGAAGGAGGA (SEQ ID NO: 348) VEGFA-Sp163 163 TGCCCTCCTCCTTCTGCCAT (SEQ ID NO: 349) VEGFA-Sp164 164 CTGCCCTCCTCCTTCTGCCA (SEQ ID NO: 350) VEGFA-Sp165 165 GGAGGGCAGAATCATCACGA (SEQ ID NO: 351) VEGFA-Sp166 166 TCATGCAGTGGTGAAGTTCA (SEQ ID NO: 352) VEGFA-Sp167 167 CTGCCATCCAATCGAGACCC (SEQ ID NO: 353) VEGFA-Sp168 168 CCATCCAATCGAGACCCTGG (SEQ ID NO: 354) VEGFA-Sp169 169 CCACCAGGGTCTCGATTGGA (SEQ ID NO: 355) VEGFA-Sp170 170 ATGTCCACCAGGGTCTCGAT (SEQ ID NO: 356) VEGFA-Sp171 171 GACCCTGGTGGACATCTTCC (SEQ ID NO: 357) VEGFA-Sp172 172 CTCCTGGAAGATGTCCACCA (SEQ ID NO: 358) VEGFA-Sp173 173 ACTCCTGGAAGATGTCCACC (SEQ ID NO: 359) VEGFA-Sp174 174 CGATCTCATCAGGGTACTCC (SEQ ID NO: 360) VEGFA-Sp175 175 AGATGTACTCGATCTCATCA (SEQ ID NO: 361) VEGFA-Sp176 176 AAGATGTACTCGATCTCATC (SEQ ID NO: 362) VEGFA-Sp177 177 CGCATCAGGGGCACACAGGA (SEQ ID NO: 363) VEGFA-Sp178 178 GCATCGCATCAGGGGCACAC (SEQ ID NO: 364) VEGFA-Sp179 179 TGTGTGCCCCTGATGCGATG (SEQ ID NO: 365) VEGFA-Sp180 180 GTGTGCCCCTGATGCGATGC (SEQ ID NO: 366) VEGFA-Sp181 181 TGTGCCCCTGATGCGATGCG (SEQ ID NO: 367) VEGFA-Sp182 182 GTGCCCCTGATGCGATGCGG (SEQ ID NO: 368) VEGFA-Sp183 183 CAGCCCCCGCATCGCATCAG (SEQ ID NO: 369) VEGFA-Sp184 184 GCAGCCCCCGCATCGCATCA (SEQ ID NO: 370) VEGFA-Sp185 185 AGCAGCCCCCGCATCGCATC (SEQ ID NO: 371) VEGFA-Sp186 186 CGGGGGCTGCTGCAATGACG (SEQ ID NO: 372) VEGFA-Sp187 187 GGGGGCTGCTGCAATGACGA (SEQ ID NO: 373) VEGFA-Sp188 188 CTGCTGCAATGACGAGGGCC (SEQ ID NO: 374) VEGFA-Sp189 189 CCTGGAGTGTGTGCCCACTG (SEQ ID NO: 375) VEGFA-Sp190 190 CCTCAGTGGGCACACACTCC (SEQ ID NO: 376) VEGFA-Sp191 191 GTGATGTTGGACTCCTCAGT (SEQ ID NO: 377) VEGFA-Sp192 192 GGTGATGTTGGACTCCTCAG (SEQ ID NO: 378) VEGFA-Sp193 193 GGAGTCCAACATCACCATGC (SEQ ID NO: 379) VEGFA-Sp194 194 GTCCAACATCACCATGCAGG (SEQ ID NO: 380) VEGFA-Sp195 195 TCCAACATCACCATGCAGGT (SEQ ID NO: 381) VEGFA-Sp196 196 GCCCACCTGCATGGTGATGT (SEQ ID NO: 382) VEGFA-Sp197 197 CCCAAAGATGCCCACCTGCA (SEQ ID NO: 383) VEGFA-Sp198 198 TCCTTCCTTTCCAGATTATG (SEQ ID NO: 384) VEGFA-Sp199 199 GAGGTTTGATCCGCATAATC (SEQ ID NO: 385) VEGFA-Sp200 200 ATGCGGATCAAACCTCACCA (SEQ ID NO: 386) VEGFA-Sp201 201 CCTCACCAAGGCCAGCACAT (SEQ ID NO: 387) VEGFA-Sp202 202 CCTATGTGCTGGCCTTGGTG (SEQ ID NO: 388) VEGFA-Sp203 203 TCTCTCCTATGTGCTGGCCT (SEQ ID NO: 389) VEGFA-Sp204 204 AGCTCATCTCTCCTATGTGC (SEQ ID NO: 390) VEGFA-Sp205 205 ATTCACATTTGTTGTGCTGT (SEQ ID NO: 391) VEGFA-Sp206 206 AGCACAACAAATGTGAATGC (SEQ ID NO: 392) VEGFA-Sp207 207 AACAAATGTGAATGCAGGTG (SEQ ID NO: 393) VEGFA-Sp208 208 TGTCTTGCTCTATCTTTCTT (SEQ ID NO: 394) VEGFA-Sp209 209 TTTTCCAGAAAATCAGTTCG (SEQ ID NO: 395) VEGFA-Sp210 210 CTTTCCTCGAACTGATTTTC (SEQ ID NO: 396) VEGFA-Sp211 211 CAGAAAATCAGTTCGAGGAA (SEQ ID NO: 397) VEGFA-Sp212 212 AGAAAATCAGTTCGAGGAAA (SEQ ID NO: 398) VEGFA-Sp213 213 ATCAGTTCGAGGAAAGGGAA (SEQ ID NO: 399) VEGFA-Sp214 214 TCAGTTCGAGGAAAGGGAAA (SEQ ID NO: 400) VEGFA-Sp215 215 CAGTTCGAGGAAAGGGAAAG (SEQ ID NO: 401) VEGFA-Sp216 216 AACGAAAGCGCAAGAAATCC (SEQ ID NO: 402) VEGFA-Sp217 217 AGAAATCCCGGTATAAGTCC (SEQ ID NO: 403) VEGFA-Sp218 218 CACGCTCCAGGACTTATACC (SEQ ID NO: 404) VEGFA-Sp219 219 ACACGCTCCAGGACTTATAC (SEQ ID NO: 405) VEGFA-Sp220 220 AAGTCCTGGAGCGTGTACGT (SEQ ID NO: 406) VEGFA-Sp221 221 GGCACCAACGTACACGCTCC (SEQ ID NO: 407) VEGFA-Sp222 222 CCCGCTGCTGTCTAATGCCC (SEQ ID NO: 408) VEGFA-Sp223 223 CCAGGGCATTAGACAGCAGC (SEQ ID NO: 409) VEGFA-Sp224 224 TCCAGGGCATTAGACAGCAG (SEQ ID NO: 410) VEGFA-Sp225 225 CTAATGCCCTGGAGCCTCCC (SEQ ID NO: 411) VEGFA-Sp226 226 TGGGGGCCAGGGAGGCTCCA (SEQ ID NO: 412) VEGFA-Sp227 227 CTGGGGGCCAGGGAGGCTCC (SEQ ID NO: 413) VEGFA-Sp228 228 GGTTGTACTGGGGGCCAGGG (SEQ ID NO: 414) VEGFA-Sp229 229 GGAGGTTGTACTGGGGGCCA (SEQ ID NO: 415) VEGFA-Sp230 230 CGGAGGTTGTACTGGGGGCC (SEQ ID NO: 416) VEGFA-Sp231 231 GCAGGCGGAGGTTGTACTGG (SEQ ID NO: 417) VEGFA-Sp232 232 TTGCCTTTTTGCAGTCCCTG (SEQ ID NO: 418) VEGFA-Sp233 233 TGCCTTTTTGCAGTCCCTGT (SEQ ID NO: 419) VEGFA-Sp234 234 CGCTCTGAGCAAGGCCCACA (SEQ ID NO: 420) VEGFA-Sp235 235 CCTGTGGGCCTTGCTCAGAG (SEQ ID NO: 421) VEGFA-Sp236 236 CCGCTCTGAGCAAGGCCCAC (SEQ ID NO: 422) VEGFA-Sp237 237 TGCTTTCTCCGCTCTGAGCA (SEQ ID NO: 423) VEGFA-Sp238 238 CAGGAACATTTACACGTCTG (SEQ ID NO: 424) VEGFA-Sp239 239 ACGCGAGTCTGTGTTTTTGC (SEQ ID NO: 425) VEGFA-Sp240 240 AAACACAGACTCGCGTTGCA (SEQ ID NO: 426) VEGFA-Sp241 241 CAGACTCGCGTTGCAAGGCG (SEQ ID NO: 427) VEGFA-Sp242 242 AGTTAAACGAACGTACTTGC (SEQ ID NO: 428) VEGFA-Sp243 243 AAACGAACGTACTTGCAGGT (SEQ ID NO: 429) VEGFA-Sp244 244 CCCTCAGATGTGACAAGCCG (SEQ ID NO: 430) VEGFA-Sp245 245 GCCTCGGCTTGTCACATCTG (SEQ ID NO: 431) VEGFA-Sp246 246 TCAGATGTGACAAGCCGAGG (SEQ ID NO: 432) VEGFA-Sp247 247 ACAAGCCGAGGCGGTGAGCC (SEQ ID NO: 433) VEGFA-Sp248 248 GCCGAGGCGGTGAGCCGGGC (SEQ ID NO: 434) VEGFA-Sp249 249 TCCTGCCCGGCTCACCGCCT (SEQ ID NO: 435)

TABLE 11 Target sequences of HIF1A gene for SpCas9 Gene No. Target sequence HIF1A-Sp1 1 TTTAAATGAGCTCCCAATGT (SEQ ID NO: 436) HIF1A-Sp2 2 GAGCTCCCAATGTCGGAGTT (SEQ ID NO: 437) HIF1A-Sp3 3 GTTTTCCAAACTCCGACATT (SEQ ID NO: 438) HIF1A-Sp4 4 TGTTTTCCAAACTCCGACAT (SEQ ID NO: 439) HIF1A-Sp5 5 AAATTTGTCTTTTTAAAAGA (SEQ ID NO: 440) HIF1A-Sp6 6 GTCTTTTTAAAAGAAGGTCT (SEQ ID NO: 441) HIF1A-Sp7 7 AAACTCAAAACCTGAAGAAT (SEQ ID NO: 442) HIF1A-Sp8 8 CTGATTTCTTCCAATTCTTC (SEQ ID NO: 443) HIF1A-Sp9 9 GAAGAAATCAGAATAGAAAA (SEQ ID NO: 444) HIF1A- 10 AAGAAATCAGAATAGAAAAT (SEQ ID NO: 445) Sp10 HIF1A- 11 ATCAGAATAGAAAATGGGTA (SEQ ID NO: 446) Sp11 HIF1A- 12 CTCGAGATGCAGCCAGATCT (SEQ ID NO: 447) Sp12 HIF1A- 13 TTCTTTACTTCGCCGAGATC (SEQ ID NO: 448) Sp13 HIF1A- 14 GAACTCACATTATGTGGAAG (SEQ ID NO: 449) Sp14 HIF1A- 15 AGATGCGAACTCACATTATG (SEQ ID NO: 450) Sp15 HIF1A- 16 TGTGAGTTCGCATCTTGATA (SEQ ID NO: 451) Sp16 HIF1A- 17 TTGATAAGGCCTCTGTGATG (SEQ ID NO: 452) Sp17 HIF1A- 18 GATGGTAAGCCTCATCACAG (SEQ ID NO: 453) Sp18 HIF1A- 19 CCATCAGCTATTTGCGTGTG (SEQ ID NO: 454) Sp19 HIF1A- 20 CCTCACACGCAAATAGCTGA (SEQ ID NO: 455) Sp20 HIF1A- 21 TTTGCGTGTGAGGAAACTTC (SEQ ID NO: 456) Sp21 HIF1A- 22 GTGAGGAAACTTCTGGATGC (SEQ ID NO: 457) Sp22 HIF1A- 23 TGTGCCCTTTTTAGGTGATT (SEQ ID NO: 458) Sp23 HIF1A- 24 TTGCTTTTATTTGAAAGCCT (SEQ ID NO: 459) Sp24 HIF1A- 25 TTTTATTTGAAAGCCTTGGA (SEQ ID NO: 460) Sp25 HIF1A- 26 AGCCTTGGATGGTTTTGTTA (SEQ ID NO: 461) Sp26 HIF1A- 27 AACCATAACAAAACCATCCA (SEQ ID NO: 462) Sp27 HIF1A- 28 GTTATGGTTCTCACAGATGA (SEQ ID NO: 463) Sp28 HIF1A- 29 TGATAATGTGAACAAATACA (SEQ ID NO: 464) Sp29 HIF1A- 30 GATAATGTGAACAAATACAT (SEQ ID NO: 465) Sp30 HIF1A- 31 CAAATACATGGGATTAACTC (SEQ ID NO: 466) Sp31 HIF1A- 32 TGTTTACAGTTTGAACTAAC (SEQ ID NO: 467) Sp32 HIF1A- 33 TACTCATCCATGTGACCATG (SEQ ID NO: 468) Sp33 HIF1A- 34 CTCATTTCCTCATGGTCACA (SEQ ID NO: 469) Sp34 HIF1A- 35 GCATTTCTCTCATTTCCTCA (SEQ ID NO: 470) Sp35 HIF1A- 36 GAAATGCTTACACACAGAAA (SEQ ID NO: 471) Sp36 HIF1A- 37 TTCATTAGGCCTTGTGAAAA (SEQ ID NO: 472) Sp37 HIF1A- 38 TCATTAGGCCTTGTGAAAAA (SEQ ID NO: 473) Sp38 HIF1A- 39 GTTCTTTACCCTTTTTCACA (SEQ ID NO: 474) Sp39 HIF1A- 40 AAGTGTACCCTAACTAGCCG (SEQ ID NO: 475) Sp40 HIF1A- 41 AGTTCTTCCTCGGCTAGTTA (SEQ ID NO: 476) Sp41 HIF1A- 42 TAGTTCTTCCTCGGCTAGTT (SEQ ID NO: 477) Sp42 HIF1A- 43 TTATGTTCATAGTTCTTCCT (SEQ ID NO: 478) Sp43 HIF1A- 44 TGAACATAAAGTCTGCAACA (SEQ ID NO: 479) Sp44 HIF1A- 45 CATAAAGTCTGCAACATGGA (SEQ ID NO: 480) Sp45 HIF1A- 46 ACACAGGTATTGCACTGCAC (SEQ ID NO: 481) Sp46 HIF1A- 47 TGGTATCATATACGTGAATG (SEQ ID NO: 482) Sp47 HIF1A- 48 ACACTGAGGTTGGTTACTGT (SEQ ID NO: 483) Sp48 HIF1A- 49 AACAGTAACCAACCTCAGTG (SEQ ID NO: 484) Sp49 HIF1A- 50 ACAGTAACCAACCTCAGTGT (SEQ ID NO: 485) Sp50 HIF1A- 51 TCTTATACCCACACTGAGGT (SEQ ID NO: 486) Sp51 HIF1A- 52 GGTTTCTTATACCCACACTG (SEQ ID NO: 487) Sp52 HIF1A- 53 GAAACCACCTATGACCTGCT (SEQ ID NO: 488) Sp53 HIF1A- 54 AGCACCAAGCAGGTCATAGG (SEQ ID NO: 489) Sp54 HIF1A- 55 ATCAGCACCAAGCAGGTCAT (SEQ ID NO: 490) Sp55 HIF1A- 56 TTCACAAATCAGCACCAAGC (SEQ ID NO: 491) Sp56 HIF1A- 57 ATATTTGATGGGTGAGGAAT (SEQ ID NO: 492) Sp57 HIF1A- 58 AATATTTGATGGGTGAGGAA (SEQ ID NO: 493) Sp58 HIF1A- 59 ATTTCAATATTTGATGGGTG (SEQ ID NO: 494) Sp59 HIF1A- 60 AAGGAATTTCAATATTTGAT (SEQ ID NO: 495) Sp60 HIF1A- 61 AAAGGAATTTCAATATTTGA (SEQ ID NO: 496) Sp61 HIF1A- 62 AGGAAAGTCTTGCTATCTAA (SEQ ID NO: 497) Sp62 HIF1A- 63 TTTCCTCAGTCGACACAGCC (SEQ ID NO: 498) Sp63 HIF1A- 64 TATCCAGGCTGTGTCGACTG (SEQ ID NO: 499) Sp64 HIF1A- 65 AATAAGAAAATTTCATATCC (SEQ ID NO: 500) Sp65 HIF1A- 66 AATTTTCTTATTGTGATGAA (SEQ ID NO: 501) Sp66 HIF1A- 67 TAACAGAATTACCGAATTGA (SEQ ID NO: 502) Sp67 HIF1A- 68 AACAGAATTACCGAATTGAT (SEQ ID NO: 503) Sp68 HIF1A- 69 TGGCTCATATCCCATCAATT (SEQ ID NO: 504) Sp69 HIF1A- 70 TATGAGCCAGAAGAACTTTT (SEQ ID NO: 505) Sp70 HIF1A- 71 GAGCGGCCTAAAAGTTCTTC (SEQ ID NO: 506) Sp71 HIF1A- 72 GATAATATTCATAAATTGAG (SEQ ID NO: 507) Sp72 HIF1A- 73 TTATGAATATTATCATGCTT (SEQ ID NO: 508) Sp73 HIF1A- 74 CTTACTATCATGATGAGTTT (SEQ ID NO: 509) Sp74 HIF1A- 75 TCCCCCCTAGTGTTTACTAA (SEQ ID NO: 510) Sp75 HIF1A- 76 TTGTCCTTTAGTAAACACTA (SEQ ID NO: 511) Sp76 HIF1A- 77 CTTGTCCTTTAGTAAACACT (SEQ ID NO: 512) Sp77 HIF1A- 78 ACTAAAGGACAAGTCACCAC (SEQ ID NO: 513) Sp78 HIF1A- 79 AAGTCACCACAGGACAGTAC (SEQ ID NO: 514) Sp79 HIF1A- 80 AAGCATCCTGTACTGTCCTG (SEQ ID NO: 515) Sp80 HIF1A- 81 TACAGGATGCTTGCCAAAAG (SEQ ID NO: 516) Sp81 HIF1A- 82 AGGATGCTTGCCAAAAGAGG (SEQ ID NO: 517) Sp82 HIF1A- 83 CCAAAAGAGGTGGATATGTC (SEQ ID NO: 518) Sp83 HIF1A- 84 CCAGACATATCCACCTCTTT (SEQ ID NO: 519) Sp84 HIF1A- 85 CAAAAGAGGTGGATATGTCT (SEQ ID NO: 520) Sp85 HIF1A- 86 GCACTGTGGTTGAGAATTCT (SEQ ID NO: 521) Sp86 HIF1A- 87 TTCACACATACAATGCACTG (SEQ ID NO: 522) Sp87 HIF1A- 88 TATGTGTGAATTACGTTGTG (SEQ ID NO: 523) Sp88 HIF1A- 89 GACACATTCTGTTTGTTGAA (SEQ ID NO: 524) Sp89 HIF1A- 90 GGACACATTCTGTTTGTTGA (SEQ ID NO: 525) Sp90 HIF1A- 91 AACAGAATGTGTCCTTAAAC (SEQ ID NO: 526) Sp91 HIF1A- 92 CTGAAGATTCAACCGGTTTA (SEQ ID NO: 527) Sp92 HIF1A- 93 TTCATATCTGAAGATTCAAC (SEQ ID NO: 528) Sp93 HIF1A- 94 TGTATCTTCTGATTCAACTT (SEQ ID NO: 529) Sp94 HIF1A- 95 CCTCTTTGACAAACTTAAGA (SEQ ID NO: 530) Sp95 HIF1A- 96 CCTTCTTAAGTTTGTCAAAG (SEQ ID NO: 531) Sp96 HIF1A- 97 ACCTGATGCTTTAACTTTGC (SEQ ID NO: 532) Sp97 HIF1A- 98 GCCAGCAAAGTTAAAGCATC (SEQ ID NO: 533) Sp98 HIF1A- 99 ACTTTGCTGGCCCCAGCCGC (SEQ ID NO: 534) Sp99 HIF1A- 100 GATTGTGTCTCCAGCGGCTG (SEQ ID NO: 535) Sp100 HIF1A- 101 TGATTGTGTCTCCAGCGGCT (SEQ ID NO: 536) Sp101 HIF1A- 102 ATGATTGTGTCTCCAGCGGC (SEQ ID NO: 537) Sp102 HIF1A- 103 AGATATGATTGTGTCTCCAG (SEQ ID NO: 538) Sp103 HIF1A- 104 ACAATCATATCTTTAGATTT (SEQ ID NO: 539) Sp104 HIF1A- 105 TCTTTAGATTTTGGCAGCAA (SEQ ID NO: 540) Sp105 HIF1A- 106 GTCATCAGTTTCTGTGTCTG (SEQ ID NO: 541) Sp106 HIF1A- 107 AACTGATGACCAGCAACTTG (SEQ ID NO: 542) Sp107 HIF1A- 108 ATGGTACTTCCTCAAGTTGC (SEQ ID NO: 543) Sp108 HIF1A- 109 AGCATTACATCATTATATAA (SEQ ID NO: 544) Sp109 HIF1A- 110 GTAATTTTTCGTTGGGTGAG (SEQ ID NO: 545) Sp110 HIF1A- 111 TGTAATTTTTCGTTGGGTGA (SEQ ID NO: 546) Sp111 HIF1A- 112 CTGTAATTTTTCGTTGGGTG (SEQ ID NO: 547) Sp112 HIF1A- 113 ATATTCTGTAATTTTTCGTT (SEQ ID NO: 548) Sp113 HIF1A- 114 TATATTCTGTAATTTTTCGT (SEQ ID NO: 549) Sp114 HIF1A- 115 AAAATTACAGAATATAAATT (SEQ ID NO: 550) Sp115 HIF1A- 116 GGCGTTTCAGCGGTGGGTAA (SEQ ID NO: 551) Sp116 HIF1A- 117 GGCTTTGGCGTTTCAGCGGT (SEQ ID NO: 552) Sp117 HIF1A- 118 TGGCTTTGGCGTTTCAGCGG (SEQ ID NO: 553) Sp118 HIF1A- 119 AAGTGGCTTTGGCGTTTCAG (SEQ ID NO: 554) Sp119 HIF1A- 120 GCACTACTTCGAAGTGGCTT (SEQ ID NO: 555) Sp120 HIF1A- 121 GGGTCAGCACTACTTCGAAG (SEQ ID NO: 556) Sp121 HIF1A- 122 CAACTTCTTGATTGAGTGCA (SEQ ID NO: 557) Sp122 HIF1A- 123 GCAACTTCTTGATTGAGTGC (SEQ ID NO: 558) Sp123 HIF1A- 124 AGAACCAAATCCAGAGTCAC (SEQ ID NO: 559) Sp124 HIF1A- 125 AGTTCCAGTGACTCTGGATT (SEQ ID NO: 560) Sp125 HIF1A- 126 AAAGAAAGTTCCAGTGACTC (SEQ ID NO: 561) Sp126 HIF1A- 127 TTTTACCATGCCCCAGATTC (SEQ ID NO: 562) Sp127 HIF1A- 128 CTGATCCTGAATCTGGGGCA (SEQ ID NO: 563) Sp128 HIF1A- 129 GGTGTCTGATCCTGAATCTG (SEQ ID NO: 564) Sp129 HIF1A- 130 AGGTGTCTGATCCTGAATCT (SEQ ID NO: 565) Sp130 HIF1A- 131 TAGGTGTCTGATCCTGAATC (SEQ ID NO: 566) Sp131 HIF1A- 132 CAGACACCTAGTCCTTCCGA (SEQ ID NO: 567) Sp132 HIF1A- 133 GTGCTTCCATCGGAAGGACT (SEQ ID NO: 568) Sp133 HIF1A- 134 TGTCTAGTCGTTCCATCGGA (SEQ ID NO: 569) Sp134 HIF1A- 135 ACTTTGTCTAGTGCTTCCAT (SEQ ID NO: 570) Sp135 HIF1A- 136 CACTAGACAAAGTTCACCTG (SEQ ID NO: 571) Sp136 HIF1A- 137 AGACAAAGTTCACCTGAGGT (SEQ ID NO: 572) Sp137 HIF1A- 138 TATATCATGACACCTACCTC (SEQ ID NO: 573) Sp138 HIF1A- 139 CAATATTCACTGGGACTATT (SEQ ID NO: 574) Sp139 HIF1A- 140 ACATAAAAACAATATTCACT (SEQ ID NO: 575) Sp140 HIF1A- 141 CACATAAAAACAATATTCAC (SEQ ID NO: 576) Sp141 HIF1A- 142 CAGTGAATATTGTTTTTATG (SEQ ID NO: 577) Sp142 HIF1A- 143 TTTTTATGTGGATAGTGATA (SEQ ID NO: 578) Sp143 HIF1A- 144 TATGGTCAATGAATTCAAGT (SEQ ID NO: 579) Sp144 HIF1A- 145 CAATGAATTCAAGTTGGAAT (SEQ ID NO: 580) Sp145 HIF1A- 146 AAAGAACCCATTTTCTACTC (SEQ ID NO: 581) Sp146 HIF1A- 147 CATATACCTGAGTAGAAAAT (SEQ ID NO: 582) Sp147 HIF1A- 148 TCATATACCTGAGTAGAAAA (SEQ ID NO: 583) Sp148 HIF1A- 149 AAAGGACACAGATTTAGACT (SEQ ID NO: 584) Sp149 HIF1A- 150 GTTAGCTCCCTATATCCCAA (SEQ ID NO: 585) Sp150 HIF1A- 151 TCATCATCCATTGGGATATA (SEQ ID NO: 586) Sp151 HIF1A- 152 GTCATCATCCATTGGGATAT (SEQ ID NO: 587) Sp152 HIF1A- 153 ACTGGAAGTCATCATCCATT (SEQ ID NO: 588) Sp153 HIF1A- 154 AACTGGAAGTCATCATCCAT (SEQ ID NO: 589) Sp154 HIF1A- 155 ACTGATCGAAGGAACGTAAC (SEQ ID NO: 590) Sp155 HIF1A- 156 TAATGGTGACAACTGATCGA (SEQ ID NO: 591) Sp156 HIF1A- 157 CTTGCGGAACTGCTTTCTAA (SEQ ID NO: 592) Sp157 HIF1A- 158 ACTTGCGCTTTCAGGGCTTG (SEQ ID NO: 593) Sp158 HIF1A- 159 TTTGAGGACTTGCGCTTTCA (SEQ ID NO: 594) Sp159 HIF1A- 160 CTTTGAGGACTTGCGCTTTC (SEQ ID NO: 595) Sp160 HIF1A- 161 AATACTGTAACTGTGCTTTG (SEQ ID NO: 596) Sp161 HIF1A- 162 GTTCTTGTATTTGAGTCTGC (SEQ ID NO: 597) Sp162 HIF1A- 163 GTAGTGGTGGCATTAGCAGT (SEQ ID NO: 598) Sp163 HIF1A- 164 AGTGGTGGCAGTGGTAGTGG (SEQ ID NO: 599) Sp164 HIF1A- 165 ATCAGTGGTGGCAGTGGTAG (SEQ ID NO: 600) Sp165 HIF1A- 166 TAATTCATCAGTGGTGGCAG (SEQ ID NO: 601) Sp166 HIF1A- 167 TGTTTTTAATTCATCAGTGG (SEQ ID NO: 602) Sp167 HIF1A- 168 CACTGTTTTTAATTCATCAG (SEQ ID NO: 603) Sp163 HIF1A- 169 AACAGTGACAAAAGACCGTA (SEQ ID NO: 604) Sp169 HIF1A- 170 ATATTTTAATGTCTTCCATA (SEQ ID NO: 605) Sp170 HIF1A- 171 TTATGTATGTGGGTAGGAGA (SEQ ID NO: 606) Sp171 HIF1A- 172 GTTTCTTTATGTATGTGGGT (SEQ ID NO: 607) Sp172 HIF1A- 173 AGTAGTTTCTTTATGTATGT (SEQ ID NO: 608) Sp173 HIF1A- 174 TAGTAGTTTCTTTATGTATG (SEQ ID NO: 609) Sp174 HIF1A- 175 ATCTCTATATGGTGATGATG (SEQ ID NO: 610) Sp175 HIF1A- 176 CGACTTTGAGTATCTCTATA (SEQ ID NO: 611) Sp176 HIF1A- 177 CATATAGAGATACTCAAAGT (SEQ ID NO: 612) Sp177 HIF1A- 178 ACAGCCTCACCAAACAGAGC (SEQ ID NO: 613) Sp178 HIF1A- 179 TTTTCCTGCTCTGTTTGGTG (SEQ ID NO: 614) Sp179 HIF1A- 180 TCACCAAACAGAGCAGGAAA (SEQ ID NO: 615) Sp180 HIF1A- 181 ACTCCTTTTCCTGCTCTGTT (SEQ ID NO: 616) Sp181 HIF1A- 182 GATAACACGTTAGGGCTTCT (SEQ ID NO: 617) Sp182 HIF1A- 183 AAGCGACAGATAACACGTTA (SEQ ID NO: 618) Sp183 HIF1A- 184 AAAGCGACAGATAACACGTT (SEQ ID NO: 619) Sp184 HIF1A- 185 TATCTGTCGCTTTGAGTCAA (SEQ ID NO: 620) Sp185 HIF1A- 186 TTTCAGAACTACAGTTCCTG (SEQ ID NO: 621) Sp186 HIF1A- 187 TTTGGATTTAGTTCTTCCTC (SEQ ID NO: 622) Sp187 HIF1A- 188 TTCTGCAAAGCTAGTATCTT (SEQ ID NO: 623) Sp188 HIF1A- 189 TGCTCAGAGAAAGCGAAAAA (SEQ ID NO: 624) Sp189 HIF1A- 190 AAGCGAAAAATGGAACATGA (SEQ ID NO: 625) Sp190 HIF1A- 191 GTAGTAGCTGCATGATCGTC (SEQ ID NO: 626) Sp191 HIF1A- 192 CAGCTACTACATCACTTTCT (SEQ ID NO: 627) Sp192 HIF1A- 193 CTTTCTTGGAAACGTGTAAA (SEQ ID NO: 628) Sp193 HIF1A- 194 ACAATTATTTTAATACCCTC (SEQ ID NO: 629) Sp194 HIF1A- 195 AAAAGAATAAACTAACCAGA (SEQ ID NO: 630) Sp195 HIF1A- 196 AAAAAGAATAAACTAACCAG (SEQ ID NO: 631) Sp196 HIF1A- 197 AGATTTAGCATGTAGACTGC (SEQ ID NO: 632) Sp197 HIF1A- 198 GATTTAGCATGTAGACTGCT (SEQ ID NO: 633) Sp198 HIF1A- 199 ATTTAGCATGTAGACTGCTG (SEQ ID NO: 634) Sp199 HIF1A- 200 TAGACTGCTGGGGCAATCAA (SEQ ID NO: 635) Sp200 HIF1A- 201 GGGCAATCAATGGATGAAAG (SEQ ID NO: 636) Sp201 HIF1A- 202 CAATCATAACTGGTCAGCTG (SEQ ID NO: 637) Sp202 HIF1A- 203 ATTAACTTCACAATCATAAC (SEQ ID NO: 638) Sp203 HIF1A- 204 GAAGTTAATGCTCCTATACA (SEQ ID NO: 639) Sp204 HIF1A- 205 AGGTTTCTGCTGCCTTGTAT (SEQ ID NO: 640) Sp205 HIF1A- 206 AGGCAGCAGAAACCTACTGC (SEQ ID NO: 641) Sp206 HIF1A- 207 GGCAGCAGAAACCTACTGCA (SEQ ID NO: 642) Sp207 HIF1A- 208 GTAATTCTTCACCCTGCAGT (SEQ ID NO: 643) Sp208 HIF1A- 209 TGAAGAATTACTCAGAGCTT (SEQ ID NO: 644) Sp209

TABLE 12 Target sequences of EPAS1 gene for SpCas9 Gene No. Target sequence EPAS1- 1 AGCGACAATGACAGCTGACA (SEQ ID NO: 645) Sp1 EPAS1- 2 CAGCTGACAAGGAGAAGAAA (SEQ ID NO: 646) Sp2 EPAS1- 3 TTCTCCACTTAGGAGTAGCT (SEQ ID NO: 647) Sp3 EPAS1- 4 CACTTAGGAGTAGCTCGGAG (SEQ ID NO: 648) Sp4 EPAS1- 5 TTAGGAGTAGCTCGGAGAGG (SEQ ID NO: 649) Sp5 EPAS1- 6 GAGTAGCTCGGAGAGGAGGA (SEQ ID NO: 650) Sp6 EPAS1- 7 AGAGGAGGAAGGAGAAGTCC (SEQ ID NO: 651) Sp7 EPAS1- 8 GAGGAGGAAGGAGAAGTCCC (SEQ ID NO: 652) Sp8 EPAS1- 9 AGAAGTCCCGGGATCCTGCG (SEQ ID NO: 653) Sp9 EPAS1- 10 CCCGGGATGCTGCGCGGTGC (SEQ ID NO: 654) Sp10 EPAS1- 11 CCGGCACCGCGCAGCATCCC (SEQ ID NO: 655) Sp11 EPAS1- 12 GCCGGCACCGCGCAGCATCC (SEQ ID NO: 656) Sp12 EPAS1- 13 GGGATGCTGCGCGGTGCCGG (SEQ ID NO: 657) Sp13 EPAS1- 14 TGCGCGGTGCCGGCGGAGCA (SEQ ID NO: 658) Sp14 EPAS1- 15 GTGCCGGCGGAGCAAGGAGA (SEQ ID NO: 659) Sp15 EPAS1- 16 CCGGCGGAGCAAGGAGACGG (SEQ ID NO: 660) Sp16 EPAS1- 17 CCTCCGTCTCCTTGCTCCGC (SEQ ID NO: 661) Sp17 EPAS1- 18 GACGGAGGTGTTCTATGAGC (SEQ ID NO: 662) Sp18 EPAS1- 19 GTGGGGCAGAGGCAGCTCAT (SEQ ID NO: 663) Sp19 EPAS1- 20 TGTGGGGCAGAGGCAGCTCA (SEQ ID NO: 664) Sp20 EPAS1- 21 GAGCTCACACTGTGGGGCAG (SEQ ID NO: 665) Sp21 EPAS1- 22 AGATGGGAGCTCACACTGTG (SEQ ID NO: 666) Sp22 EPAS1- 23 CAGATGGGAGCTCACACTGT (SEQ ID NO: 667) Sp23 EPAS1- 24 CCACAGTGTGAGCTCCCATC (SEQ ID NO: 668) Sp24 EPAS1- 25 CCAGATGGGAGCTCACACTG (SEQ ID NO: 669) Sp25 EPAS1- 26 TGTGAGCTCCCATCTGGACA (SEQ ID NO: 670) Sp26 EPAS1- 27 GATGGAGGCCTTGTCCAGAT (SEQ ID NO: 671) Sp27 EPAS1- 28 TGATGGAGGCCTTGTCCAGA (SEQ ID NO: 672) Sp28 EPAS1- 29 CAAGGCCTCCATCATGCGAC (SEQ ID NO: 673) Sp29 EPAS1- 30 GATTGCCAGTCGCATGATGG (SEQ ID NO: 674) Sp30 EPAS1- 31 GCTGATTGCCAGTCGCATGA (SEQ ID NO: 675) Sp31 EPAS1- 32 AGAGGAGCTTGTGTGTTCGC (SEQ ID NO: 676) Sp32 EPAS1- 33 ACACACAAGCTCCTCTCCTC (SEQ ID NO: 677) Sp33 EPAS1- 34 CAAGCTCCTCTCCTCAGGTA (SEQ ID NO: 678) Sp34 EPAS1- 35 TGCTGGCCTTACCTGAGGAG (SEQ ID NO: 679) Sp35 EPAS1- 36 GAGCCTGCTGGCCTTACCTG (SEQ ID NO: 680) Sp36 EPAS1- 37 GACTCGTTTTCAGAGCAAAC (SEQ ID NO: 681) Sp37 EPAS1- 38 CTGCTGGTCAGCTTCGGCTT (SEQ ID NO: 682) Sp38 EPAS1- 39 AGCCGAAGCTGACCAGCAGA (SEQ ID NO: 683) Sp39 EPAS1- 40 GTCCATCTGCTGGTCAGCTT (SEQ ID NO: 684) Sp40 EPAS1- 41 GGTACAAGTTGTCCATCTGC (SEQ ID NO: 685) Sp41 EPAS1- 42 CAACTTGTACCTGAAAGCCT (SEQ ID NO: 686) Sp42 EPAS1- 43 CTTGTACCTGAAAGCCTTGG (SEQ ID NO: 687) Sp43 EPAS1- 44 TTGTACCTGAAAGCCTTGGA (SEQ ID NO: 688) Sp44 EPAS1- 45 TGAAACCCTCCAAGGCTTTC (SEQ ID NO: 689) Sp45 EPAS1- 46 CACGGCAATGAAACCCTCCA (SEQ ID NO: 690) Sp46 EPAS1- 47 CTTGGAGGGTTTCATTGCCG (SEQ ID NO: 691) Sp47 EPAS1- 48 ATTGCCGTGGTGACCCAAGA (SEQ ID NO: 692) Sp48 EPAS1- 49 GTCGCCATCTTGGGTCACCA (SEQ ID NO: 693) Sp49 EPAS1- 50 AAAGATCATGTCGCCATCTT (SEQ ID NO: 694) Sp50 EPAS1- 51 GAAAGATCATGTCGCCATCT (SEQ ID NO: 695) Sp51 EPAS1- 52 AGAAAACATCAGCAAGTTCA (SEQ ID NO: 696) Sp52 EPAS1- 53 GAAAACATCAGCAAGTTCAT (SEQ ID NO: 697) Sp53 EPAS1- 54 CAAGTTCATGGGACTTACAC (SEQ ID NO: 698) Sp54 EPAS1- 55 TTGAAACAGGTGGAGCTAAC (SEQ ID NO: 699) Sp55 EPAS1- 56 CACTCATCCCTGCGACCATG (SEQ ID NO: 700) Sp56 EPAS1- 57 CGAATCTCCTCATGGTCGCA (SEQ ID NO: 701) Sp57 EPAS1- ACGAATCTCCTCATGGTCGC (SEQ ID NO: 702) Sp58 EPAS1- 59 GGTTCTCACGAATCTCCTCA (SEQ ID NO: 703) Sp59 EPAS1- 60 GAGAACCTGAGTCTCAAAAA (SEQ ID NO: 704) Sp60 EPAS1- 61 GGATACCATTTTTGAGACTC (SEQ ID NO: 705) Sp61 EPAS1- 62 ATCCTTCCACATCCAGGCTC (SEQ ID NO: 706) Sp62 EPAS1- 63 CCACATCCAGGCTCTGGTTT (SEQ ID NO: 707) Sp63 EPAS1- 64 CACATCCAGGCTCTGGTTTT (SEQ ID NO: 708) Sp64 EPAS1- 65 TTTTTCCCAAAACCAGAGCC (SEQ ID NO: 709) Sp65 EPAS1- 66 GCAAAGACATGTCCACAGAG (SEQ ID NO: 710) Sp66 EPAS1- 67 CAAAGACATGTCCACAGAGC (SEQ ID NO: 711) Sp67 EPAS1- 68 CATGAAGAAGTCCCGCTCTG (SEQ ID NO: 712) Sp68 EPAS1- 69 CAGAGCGGGACTTCTTCATG (SEQ ID NO: 713) Sp69 EPAS1- 70 CTTCATGAGGATGAAGTGCA (SEQ ID NO: 714) Sp70 EPAS1- 71 AAGTGCACGGTCACCAACAG (SEQ ID NO: 715) Sp71 EPAS1- 72 GTTGACAGTACGGCCTCTGT (SEQ ID NO: 716) Sp72 EPAS1- 73 CTGACTTGAGGTTGACAGTA (SEQ ID NO: 717) Sp73 EPAS1- 74 TCAACCTCAAGTCAGCCACC (SEQ ID NO: 718) Sp74 EPAS1- 75 CCTCAAGTCAGCCACCTGGA (SEQ ID NO: 719) Sp75 EPAS1- 76 CCTTCCAGGTGGCTGACTTG (SEQ ID NO: 720) Sp76 EPAS1- 77 AAGTCAGCCACCTGGAAGGT (SEQ ID NO: 721) Sp77 EPAS1- 78 AGTCAGCCACCTGGAAGGTA (SEQ ID NO: 722) Sp78 EPAS1- 79 ATGTTGCCCTACCTTCCAGG (SEQ ID NO: 723) Sp79 EPAS1- 80 CTGATGTTGCCCTACCTTCC (SEQ ID NO: 724) Sp80 EPAS1- 81 GTCTCAGGTCTTGCACTGCA (SEQ ID NO: 725) Sp81 EPAS1- 82 TCTCAGGTCTTGCACTGCAC (SEQ ID NO: 726) Sp82 EPAS1- 83 GGTCTTGCACTGCACGGGCC (SEQ ID NO: 727) Sp83 EPAS1- 84 AGTTGTTGTAGACTTTCACC (SEQ ID NO: 728) Sp84 EPAS1- 85 CACACAGACTATTGTGAGGA (SEQ ID NO: 729) Sp85 EPAS1- 86 CCTCCTCACAATAGTCTGTG (SEQ ID NO: 730) Sp86 EPAS1- 87 CCACACAGACTATTGTGAGG (SEQ ID NO: 731) Sp87 EPAS1- 88 TAGCCACACAGACTATTGTG (SEQ ID NO: 732) Sp88 EPAS1- 89 CAATAGTCTGTGTGGCTACA (SEQ ID NO: 733) Sp89 EPAS1- 90 ATGATGAGGCAGGACAGCAG (SEQ ID NO: 734) Sp90 EPAS1- 91 GATGATGAGGCAGGACAGCA (SEQ ID NO: 735) Sp91 EPAS1- 92 TGATGATGAGGCAGGACAGC (SEQ ID NO: 736) Sp92 EPAS1- 93 TTCACACATGATGATGAGGC (SEQ ID NO: 737) Sp93 EPAS1- 94 TTGGTTCACACATGATGATG (SEQ ID NO: 738) Sp94 EPAS1- 95 ATGTGGGATGGGTGCTGGAT (SEQ ID NO: 739) Sp95 EPAS1- 96 AATCCAGCACCCATCCCACA (SEQ ID NO: 740) Sp96 EPAS1- 97 TGTCCATGTGGGATGGGTGC (SEQ ID NO: 741) Sp97 EPAS1- 98 GGGGGATGTCCATGTGGGAT (SEQ ID NO: 742) Sp98 EPAS1- 99 AGGGGGATGTCCATGTGGGA (SEQ ID NO: 743) Sp99 EPAS1- 100 ATCCCACATGGACATCCCCC (SEQ ID NO: 744) Sp100 EPAS1- 101 ATCCAGGGGGATGTCCATGT (SEQ ID NO: 745) Sp101 EPAS1- 102 TATCCAGGGGGATGTCCATG (SEQ ID NO: 746) Sp102 EPAS1- 103 GGAAGGTCTTGCTATCCAGG (SEQ ID NO: 747) Sp103 EPAS1- 104 AGGAAGGTCTTGCTATCCAG (SEQ ID NO: 748) Sp104 EPAS1- 105 CAGGAAGGTCTTGCTATCCA (SEQ ID NO: 749) Sp105 EPAS1- 106 TCAGGAAGGTCTTGCTATCC (SEQ ID NO: 750) Sp106 EPAS1- 107 CATGCTGTGGCGGCTCAGGA (SEQ ID NO: 751) Sp107 EPAS1- 108 CTTCCTGAGCCGCCACAGCA (SEQ ID NO: 752) Sp108 EPAS1- 109 TGTCCATGCTGTGGCGGCTC (SEQ ID NO: 753) Sp109 EPAS1- 110 ACTTCATGTCCATGCTGTGG (SEQ ID NO: 754) Sp110 EPAS1- 111 TGAACTTCATGTCCATGCTG (SEQ ID NO: 755) Sp111 EPAS1- 112 AGTTCACCTACTGTGATGAC (SEQ ID NO: 756) Sp112 EPAS1- 113 CACCTACTGTGATGACAGGT (SEQ ID NO: 757) Sp113 EPAS1- 114 ACCTACTGTGATGACAGGTA (SEQ ID NO: 758) Sp114 EPAS1- 115 CCCCTACCTGTCATCACAGT (SEQ ID NO: 759) Sp115 EPAS1- 116 CTCAGAATCACAGAACTGAT (SEQ ID NO: 760) Sp116 EPAS1- 117 ACTGATTGGTTACCACCCTG (SEQ ID NO: 761) Sp117 EPAS1- 118 TACCACCCTGAGGAGCTGCT (SEQ ID NO: 762) Sp118 EPAS1- 119 GGCCAAGCAGCTCCTCAGGG (SEQ ID NO: 763) Sp119 EPAS1- 120 AGCGGCCAAGCAGCTCCTCA (SEQ ID NO: 764) Sp120 EPAS1- 121 GAGCGGCCAAGCAGCTCCTC (SEQ ID NO: 765) Sp121 EPAS1- 122 GGTAGAATTCATAGGCTGAG (SEQ ID NO: 766) Sp122 EPAS1- 123 TAGCGCATGGTAGAATTCAT (SEQ ID NO: 767) Sp123 EPAS1- 124 TGTTCTCGGAGTCTAGCGCA (SEQ ID NO: 768) Sp124 EPAS1- 125 GTGACTCTTGGTCATGTTCT (SEQ ID NO: 769) Sp125 EPAS1- 126 CTCACAGTTCTGGTGACTCT (SEQ ID NO: 770) Sp126 EPAS1- 127 ACTCCTGGAACTCACAGTTC (SEQ ID NO: 771) Sp127 EPAS1- 128 TCCTCCCCTAGTGTGCACCA (SEQ ID NO: 772) Sp128 EPAS1- 129 CCTCCCCTAGTGTGCACCAA (SEQ ID NO: 773) Sp129 EPAS1- 130 CTGACCCTTGGTGCACACTA (SEQ ID NO: 774) Sp130 EPAS1- 131 CCTAGTGTGCACCAAGGGTC (SEQ ID NO: 775) Sp131 EPAS1- 132 CCTGACCCTTGGTGCACACT (SEQ ID NO: 776) Sp132 EPAS1- 133 ACCAAGGGTCAGGTAGTAAG (SEQ ID NO: 777) Sp133 EPAS1- 134 GCCACTTACTACCTGACCCT (SEQ ID NO: 778) Sp134 EPAS1- 135 AGGTAGTAAGTGGCCAGTAC (SEQ ID NO: 779) Sp135 EPAS1- 136 GCTTTGCGAGCATCCGGTAC (SEQ ID NO: 780) Sp136 EPAS1- 137 TACCGGATGCTCGCAAAGCA (SEQ ID NO: 781) Sp137 EPAS1- 138 ACCGGATGCTCGCAAAGCAT (SEQ ID NO: 782) Sp138 EPAS1- 139 CCGGATGCTCGCAAAGCATG (SEQ ID NO: 783) Sp139 EPAS1- 140 CCCCATGCTTTGCGAGCATC (SEQ ID NO: 784) Sp140 EPAS1- 141 CGGATGCTCGCAAAGCATGG (SEQ ID NO: 785) Sp141 EPAS1- 142 CAAAGCATGGGGGCTACGTG (SEQ ID NO: 786) Sp142 EPAS1- 143 GCATGGGGGCTACGTGTGGC (SEQ ID NO: 787) Sp143 EPAS1- 144 CTACGTGTGGCTGGAGACCC (SEQ ID NO: 788) Sp144 EPAS1- 145 TACGTGTGGCTGGAGACCCA (SEQ ID NO: 789) Sp145 EPAS1- 146 ACGTGTGGCTGGAGACCCAG (SEQ ID NO: 790) Sp146 EPAS1- 147 GTGGCTGGAGACCCAGGGGA (SEQ ID NO: 791) Sp147 EPAS1- 148 GTTGTAGATGACCGTCCCCT (SEQ ID NO: 792) Sp148 EPAS1- 149 GGTTGTAGATGACCGTCCCC (SEQ ID NO: 793) Sp149 EPAS1- 150 ACTGGGGCTGCAGGTTGCGA (SEQ ID NO: 794) Sp150 EPAS1- 151 CACTGGGGCTGCAGGTTGCG (SEQ ID NO: 795) Sp151 EPAS1- 152 ACATGATGCACTGGGGCTGC (SEQ ID NO: 796) Sp152 EPAS1- 153 TTGACACACATGATGCACTG (SEQ ID NO: 797) Sp153 EPAS1- 154 GTTGACACACATGATGCACT (SEQ ID NO: 798) Sp154 EPAS1- 155 AGTTGACACACATGATGCAC (SEQ ID NO: 799) Sp155 EPAS1- 156 TGTGTGTCAACTACGTCCTG (SEQ ID NO: 800) Sp156 EPAS1- 157 AGCCCTCACATGCTTACCTC (SEQ ID NO: 801) Sp157 EPAS1- 158 TGAGATTGAGAAGAATGACG (SEQ ID NO: 802) Sp158 EPAS1- 159 GAATGACGTGGTGTTCTCCA (SEQ ID NO: 803) Sp159 EPAS1- 160 CAGGGATTCAGTCTGGTCCA (SEQ ID NO: 804) Sp160 EPAS1- 161 GCTTGAACAGGGATTCAGTC (SEQ ID NO: 805) Sp161 EPAS1- 162 CATCAGGTGGGGCTTGAACA (SEQ ID NO: 806) Sp162 EPAS1- 163 CCTGTTCAAGCCCCACCTGA (SEQ ID NO: 807) Sp163 EPAS1- 164 CCATCAGGTGGGGCTTGAAC (SEQ ID NO: 808) Sp164 EPAS1- 165 CTGTTCATGGCCATCAGGTG (SEQ ID NO: 809) Sp165 EPAS1- 166 GCTGTTCATGGCCATCAGGT (SEQ ID NO: 810) Sp166 EPAS1- 167 TGCTGTTCATGGCCATCAGG (SEQ ID NO: 811) Sp167 EPAS1- 168 AGATGCTGTTCATGGCCATC (SEQ ID NO: 812) Sp168 EPAS1- 169 GCTATCAAAGATGCTGTTCA (SEQ ID NO: 813) Sp169 EPAS1- 170 AACAGCATCTTTGATAGCAG (SEQ ID NO: 814) Sp170 EPAS1- 171 CATCTTTGATAGCAGTGGCA (SEQ ID NO: 815) Sp171 EPAS1- 172 ATCTTTGATAGCAGTGGCAA (SEQ ID NO: 816) Sp172 EPAS1- 173 TCTTTGATAGCAGTGGCAAG (SEQ ID NO: 817) Sp173 EPAS1- 174 CTTTGATAGCAGTGGCAAGG (SEQ ID NO: 818) Sp174 EPAS1- 175 CTTCCTATTCACCAAGCTAA (SEQ ID NO: 819) Sp175 EPAS1- 176 CCTATTCACCAAGCTAAAGG (SEQ ID NO: 820) Sp176 EPAS1- 177 CCTCCTTTAGCTTGGTGAAT (SEQ ID NO: 821) Sp177 EPAS1- 178 CTCGGGCTCCTCCTTTAGCT (SEQ ID NO: 822) Sp178 EPAS1- 179 CAAGCTAAAGGAGGAGCCCG (SEQ ID NO: 823) Sp179 EPAS1- 180 AAAGGAGGAGCCCGAGGAGC (SEQ ID NO: 824) Sp180 EPAS1- 181 GCCCGAGGAGCTGGCCCAGC (SEQ ID NO: 825) Sp181 EPAS1- 182 GCCAGCTGGGCCAGCTCCTC (SEQ ID NO: 826) Sp182 EPAS1- 183 AGCCAGCTGGGCCAGCTCCT (SEQ ID NO: 827) Sp183 EPAS1- 184 GCCCAGCTGGCTCCCACCCC (SEQ ID NO: 828) Sp184 EPAS1- 185 TCCTGGGGTGGGAGCCAGCT (SEQ ID NO: 829) Sp185 EPAS1- 186 CTCCTGGGGTGGGAGCCAGC (SEQ ID NO: 830) Sp186 EPAS1- 187 ATGATGGCGTCTCCTGGGGT (SEQ ID NO: 831) Sp187 EPAS1- 188 GATGATGGCGTCTCCTGGGG (SEQ ID NO: 832) Sp188 EPAS1- 189 AGAGATGATGGCGTCTCCTG (SEQ ID NO: 833) Sp189 EPAS1- 190 GAGAGATGATGGCGTCTCCT (SEQ ID NO: 834) Sp190 EPAS1- 191 AGAGAGATGATGGCGTCTCC (SEQ ID NO: 835) Sp191 EPAS1- 192 AGGAGACGCCATCATCTCTC (SEQ ID NO: 836) Sp192 EPAS1- 193 GCCATCATCTCTCTGGATTT (SEQ ID NO: 837) Sp193 EPAS1- 194 ACCGAAATCCAGAGAGATGA (SEQ ID NO: 838) Sp194 EPAS1- 195 ATCATCTCTCTGGATTTCGG (SEQ ID NO: 839) Sp195 EPAS1- 196 TCATCTCTCTGGATTTCGGT (SEQ ID NO: 840) Sp196 EPAS1- 197 CTCGAAGTTCTGATTCCCTG (SEQ ID NO: 841) Sp197 EPAS1- 198 CACAGGGAATCAGAACTTCG (SEQ ID NO: 842) Sp198 EPAS1- 199 TTCGAGGAGTCCTCAGCCTA (SEQ ID NO: 843) Sp199 EPAS1- 200 GGAGTCCTCAGCCTATGGCA (SEQ ID NO: 844) Sp200 EPAS1- 201 GATGGCCTTGCCATAGGCTG (SEQ ID NO: 845) Sp201 EPAS1- 202 GGGCAGGATGGCCTTGCCAT (SEQ ID NO: 846) Sp202 EPAS1- 203 TGGCTGGCTCGGGGGCAGGA (SEQ ID NO: 847) Sp203 EPAS1- 204 TCCTGCCCCCGAGCCAGCCA (SEQ ID NO: 848) Sp204 EPAS1- 205 CCTGCCCCCGAGCCAGCCAT (SEQ ID NO: 849) Sp205 EPAS1- 206 CCCATGGCTGGCTCGGGGGC (SEQ ID NO: 850) Sp206 EPAS1- 207 GTGGCCCATGGCTGGCTCGG (SEQ ID NO: 851) Sp207 EPAS1- 208 CGTGGCCCATGGCTGGCTCG (SEQ ID NO: 852) Sp208 EPAS1- 209 CCCGAGCCAGCCATGGGCCA (SEQ ID NO: 853) Sp209 EPAS1- 210 CCGTGGCCCATGGCTGGCTC (SEQ ID NO: 854) Sp210 EPAS1- 211 TCCGTGGCCCATGGCTGGCT (SEQ ID NO: 855) Sp211 EPAS1- 212 TCAACTCCGTGGCCCATGGC (SEQ ID NO: 856) Sp212 EPAS1- 213 AGCCATGGGCCACGGAGTTG (SEQ ID NO: 857) Sp213 EPAS1- 214 CTCCTCAACTCCGTGGCCCA (SEQ ID NO: 858) Sp214 EPAS1- 215 GCTGTGGCTCCTCAACTCCG (SEQ ID NO: 859) Sp215 EPAS1- 216 GAGCCACAGCACCCAGAGCG (SEQ ID NO: 860) Sp216 EPAS1- 217 CAGCCTCGCTCTGGGTGCTG (SEQ ID NO: 861) Sp217 EPAS1- 218 CACAGCACCCAGAGCGAGGC (SEQ ID NO: 862) Sp218 EPAS1- 219 ACAGCACCCAGAGCGAGGCT (SEQ ID NO: 863) Sp219 EPAS1- 220 CAGGCTCCCAGCCTCGCTCT (SEQ ID NO: 864) Sp220 EPAS1- 221 GCAGGCTCCCAGCCTCGCTC (SEQ ID NO: 865) Sp221 EPAS1- 222 GGGGCACGGTGAAGGCAGGC (SEQ ID NO: 866) Sp222 EPAS1- 223 GCCTGCCTTCACCGTGCCCC (SEQ ID NO: 867) Sp223 EPAS1- 224 GCCTGGGGCACGGTGAAGGC (SEQ ID NO: 868) Sp224 EPAS1- 225 AGCTGCCTGGGGCACGGTGA (SEQ ID NO: 869) Sp225 EPAS1- 226 CGGGGCAGCTGCCTGGGGCA (SEQ ID NO: 870) Sp226 EPAS1- 227 CGTGCCCCAGGCAGCTGCCC (SEQ ID NO: 871) Sp227 EPAS1- 228 GTGCCCCAGGCAGCTGCCCC (SEQ ID NO: 872) Sp228 EPAS1- 229 CTGCCCGGGGCAGCTGCCTG (SEQ ID NO: 873) Sp229 EPAS1- 230 GCTGCCCGGGGCAGCTGCCT (SEQ ID NO: 874) Sp230 EPAS1- 231 TGCTGCCCGGGGCAGCTGCC (SEQ ID NO: 875) Sp231 EPAS1- 232 ACTGGGGGTGGTGCTGCCCG (SEQ ID NO: 876) Sp232 EPAS1- 233 CACTGGGGGTGGTGCTGCCC (SEQ ID NO: 877) Sp233 EPAS1- 234 GCACTGGGGGTGGTGCTGCC (SEQ ID NO: 878) Sp234 EPAS1- 235 GCTGCTGGTGGCACTGGGGG (SEQ ID NO: 879) Sp235 EPAS1- 236 GCTGCTGCTGGTGGCACTGG (SEQ ID NO: 880) Sp236 EPAS1- 237 TGCTGCTGCTGGTGGCACTG (SEQ ID NO: 881) Sp237 EPAS1- 238 CTGCTGCTGCTGGTGGCACT (SEQ ID NO: 882) Sp238 EPAS1- 239 GCTGCTGCTGCTGGTGGCAC (SEQ ID NO: 883) Sp239 EPAS1- 240 GCAGCTGCTGCTGCTGCTGG (SEQ ID NO: 884) Sp240 EPAS1- 241 CAGCAGCAGCAGCTGCTCCA (SEQ ID NO: 885) Sp241 EPAS1- 242 TCTTCAGGGCTATTGGGCTA (SEQ ID NO: 886) Sp242 EPAS1- 243 GTCTTCAGGGCTATTGGGCT (SEQ ID NO: 887) Sp243 EPAS1- 244 TAATAGTCTTCAGGGCTATT (SEQ ID NO: 888) Sp244 EPAS1- 245 GTAATAGTCTTCAGGGCTAT (SEQ ID NO: 889) Sp245 EPAS1- 246 AAGATGTGTAATAGTCTTCA (SEQ ID NO: 890) Sp246 EPAS1- 247 AAAGATGTGTAATAGTCTTC (SEQ ID NO: 891) Sp247 EPAS1- 248 TGAAGACTATTACACATCTT (SEQ ID NO: 892) Sp248 EPAS1- 249 TCTCAATCACTTCAATCTTC (SEQ ID NO: 893) Sp249 EPAS1- 250 GATTGAGAAGCTCTTCGCCA (SEQ ID NO: 894) Sp250 EPAS1- 251 GCTCTTCGCCATGGACACAG (SEQ ID NO: 895) Sp251 EPAS1- 252 CGCCATGGACACAGAGGCCA (SEQ ID NO: 896) Sp252 EPAS1- 253 GTCCTTGGCCTCTGTGTCCA (SEQ ID NO: 897) Sp253 EPAS1- 254 CTGGGTACTGCATTGGTCCT (SEQ ID NO: 898) Sp254 EPAS1- 255 CAAGGACCAATGCAGTACCC (SEQ ID NO: 899) Sp255 EPAS1- 256 CATCTACCTGGGTACTGCAT (SEQ ID NO: 900) Sp256 EPAS1- 257 AGCTCATTGAAATCCGTCTG (SEQ ID NO: 901) Sp257 EPAS1- 258 TCAGACGGATTTCAATGAGC (SEQ ID NO: 902) Sp258 EPAS1- 259 GGATTTCAATGAGCTGGACT (SEQ ID NO: 903) Sp259 EPAS1- 260 TGAGCTGGACTTGGAGACAC (SEQ ID NO: 904) Sp260 EPAS1- 261 ACTGGCACCCTATATCCCCA (SEQ ID NO: 905) Sp261 EPAS1- 262 GCACCCTATATCCCCATGGA (SEQ ID NO: 906) Sp262 EPAS1- 263 CACCCTATATCCCCATGGAC (SEQ ID NO: 907) Sp263 EPAS1- 264 ACCCTATATCCCCATGGACG (SEQ ID NO: 908) Sp264 EPAS1- 265 TCCCCGTCCATGGGGATATA (SEQ ID NO: 909) Sp265 EPAS1- 266 TTCCCCGTCCATGGGGATAT (SEQ ID NO: 910) Sp266 EPAS1- 267 GGAAGTCTTCCCCGTCCATG (SEQ ID NO: 911) Sp267 EPAS1- 268 TGGAAGTCTTCCCCGTCCAT (SEQ ID NO: 912) Sp268 EPAS1- 269 CTGGAAGTCTTCCCCGTCCA (SEQ ID NO: 913) Sp269 EPAS1- 270 CGGGGCAGATGGGGCTTAGC (SEQ ID NO: 914) Sp270 EPAS1- 271 GCTAAGCCCCATCTGCCCCG (SEQ ID NO: 915) Sp271 EPAS1- 272 GCCCCATCTGCCCCGAGGAG (SEQ ID NO: 916) Sp272 EPAS1- 273 GCCGCTCCTCGGGGCAGATG (SEQ ID NO: 917) Sp273 EPAS1- 274 AGCCGCTCCTCGGGGCAGAT (SEQ ID NO: 918) Sp274 EPAS1- 275 GAGCCGCTCCTCGGGGCAGA (SEQ ID NO: 919) Sp275 EPAS1- 276 CTGCCCCGAGGAGCGGCTCT (SEQ ID NO: 920) Sp276 EPAS1- 277 CCCCGAGGAGCGGCTCTTGG (SEQ ID NO: 921) Sp277 EPAS1- 278 CCGCCAAGAGCCGCTCCTCG (SEQ ID NO: 922) Sp278 EPAS1- 279 TCCGCCAAGAGCCGCTCCTC (SEQ ID NO: 923) Sp279 EPAS1- 280 CTCCGCCAAGAGCCGCTCCT (SEQ ID NO: 924) Sp280 EPAS1- 281 AGTGCTGGGGGGTGGACTGT (SEQ ID NO: 925) Sp281 EPAS1- 282 CAGTGCTGGGGGGTGGACTG (SEQ ID NO: 926) Sp282 EPAS1- 283 ACTGAAGCAGTGCTGGGGGG (SEQ ID NO: 927) Sp283 EPAS1- 284 GGCACTGAAGCAGTGCTGGG (SEQ ID NO: 928) Sp284 EPAS1- 285 TGGCACTGAAGCAGTGCTGG (SEQ ID NO: 929) Sp285 EPAS1- 286 ATGGCACTGAAGCAGTGCTG (SEQ ID NO: 930) Sp286 EPAS1- 287 CATGGCACTGAAGCAGTGCT (SEQ ID NO: 931) Sp287 EPAS1- 288 TCATGGCACTGAAGCAGTGC (SEQ ID NO: 932) Sp288 EPAS1- 289 TGGCTGGAAGATGTTTGTCA (SEQ ID NO: 933) Sp289 EPAS1- 290 GACAAACATCTTCCAGCCAC (SEQ ID NO: 934) Sp290 EPAS1- 291 GGGCTACAGGGGCCAGTGGC (SEQ ID NO: 935) Sp291 EPAS1- 292 TGCGGGGCTACAGGGGCCAG (SEQ ID NO: 936) Sp292 EPAS1- 293 GGGACTGTGCGGGGCTACAG (SEQ ID NO: 937) Sp293 EPAS1- 294 AGGGACTGTGCGGGGCTACA (SEQ ID NO: 938) Sp294 EPAS1- 295 AAGGGACTGTGCGGGGCTAC (SEQ ID NO: 939) Sp295 EPAS1- 296 CAGGAGGAAGGGACTGTGCG (SEQ ID NO: 940) Sp296 EPAS1- 297 CCCGCACAGTCCCTTCCTCC (SEQ ID NO: 941) Sp297 EPAS1- 298 CCAGGAGGAAGGGACTGTGC (SEQ ID NO: 942) Sp298 EPAS1- 299 TCCAGGAGGAAGGGACTGTG (SEQ ID NO: 943) Sp299 EPAS1- 300 TGAAACTTGTCCAGGAGGAA (SEQ ID NO: 944) Sp300 EPAS1- 301 CTGAAACTTGTCCAGGAGGA (SEQ ID NO: 945) Sp301 EPAS1- 302 GCTGCTGAAACTTGTCCAGG (SEQ ID NO: 946) Sp302 EPAS1- 303 GCTGCTGCTGAAACTTGTCC (SEQ ID NO: 947) Sp303 EPAS1- 304 GGACAAGTTTCAGCAGCAGC (SEQ ID NO: 948) Sp304 EPAS1- 305 AGAAGACAGAGCCCGAGCAC (SEQ ID NO: 949) Sp305 EPAS1- 306 GAGGACATGGGCCGGTGCTC (SEQ ID NO: 950) Sp306 EPAS1- 307 GGAGGACATGGGCCGGTGCT (SEQ ID NO: 951) Sp387 EPAS1- 308 AGAAGATGGAGGACATGGGC (SEQ ID NO: 952) Sp308 EPAS1- 309 TCAAAGAAGATGGAGGACAT (SEQ ID NO: 953) Sp309 EPAS1- 310 ATCAAAGAAGATGGAGGACA (SEQ ID NO: 954) Sp310 EPAS1- 311 TCCTCCATCTTCTTTGATGC (SEQ ID NO: 955) Sp311 EPAS1- 312 TCCGGCATCAAAGAAGATGG (SEQ ID NO: 956) Sp312 EPAS1- 313 GCTTCCGGCATCAAAGAAGA (SEQ ID NO: 957) Sp313 EPAS1- 314 TGGCAGGGATGCTTTGCTTC (SEQ ID NO: 958) Sp314 EPAS1- 315 GCATCCCTGCCACCGTGCTG (SEQ ID NO: 959) Sp315 EPAS1- 316 CTGGCCACAGCACGGTGGCA (SEQ ID NO: 960) Sp316 EPAS1- 317 CCTGCCACCGTGCTGTGGCC (SEQ ID NO: 961) Sp317 EPAS1- 318 CCTGGCCACAGCACGGTGGC (SEQ ID NO: 962) Sp318 EPAS1- 319 CTGGCCTGGCCACAGCACGG (SEQ ID NO: 963) Sp319 EPAS1- 320 GTGCTGGCCTGGCCACAGCA (SEQ ID NO: 964) Sp320 EPAS1- 321 AAGAGAGAGGGGTGCTGGCC (SEQ ID NO: 965) Sp321 EPAS1- 322 CATGGAAGAGAGAGGGGTGC (SEQ ID NO: 966) Sp322 EPAS1- 323 CAGCACCCCTCTCTCTTCCA (SEQ ID NO: 967) Sp323 EPAS1- 324 AGCACCCCTCTCTCTTCCAT (SEQ ID NO: 968) Sp324 EPAS1- 325 GCACCCCTCTCTCTTCCATG (SEQ ID NO: 969) Sp325 EPAS1- 326 CACCCCTCTCTCTTCCATGG (SEQ ID NO: 970) Sp326 EPAS1- 327 ACCCCTCTCTCTTCCATGGG (SEQ ID NO: 971) Sp327 EPAS1- 328 GCCCCCCATGGAAGAGAGAG (SEQ ID NO: 972) Sp328 EPAS1- 329 TGCCCCCCATGGAAGAGAGA (SEQ ID NO: 973) Sp329 EPAS1- 330 CTGCCCCCCATGGAAGAGAG (SEQ ID NO: 974) Sp330 EPAS1- 331 GGTATTGGATCTGCCCCCCA (SEQ ID NO: 975) Sp331 EPAS1- 332 GGGGCAGATCCAATACCCAG (SEQ ID NO: 976) Sp332 EPAS1- 333 ATCTGGGGGCCACTGGGTAT (SEQ ID NO: 977) Sp333 EPAS1- 334 TGGTGGATCTGGGGGCCACT (SEQ ID NO: 978) Sp334 EPAS1- 335 ATGGTGGATCTGGGGGCCAC (SEQ ID NO: 979) Sp335 EPAS1- 336 AAATGTAATGGTGGATCTGG (SEQ ID NO: 980) Sp336 EPAS1- 337 AAAATGTAATGGTGGATCTG (SEQ ID NO: 981) Sp337 EPAS1- 338 CAAAATGTAATGGTGGATCT (SEQ ID NO: 982) Sp338 EPAS1- 339 CCAGATCCACCATTACATTT (SEQ ID NO: 983) Sp339 EPAS1- 340 CCAAAATGTAATGGTGGATC (SEQ ID NO: 984) Sp340 EPAS1- 341 CAGATCCACCATTACATTTT (SEQ ID NO: 985) Sp341 EPAS1- 342 GTGGGCCCAAAATGTAATGG (SEQ ID NO: 986) Sp342 EPAS1- 343 TTTGTGGGCCCAAAATGTAA (SEQ ID NO: 987) Sp343 EPAS1- 344 TACATTTTGGGCCCACAAAG (SEQ ID NO: 988) Sp344 EPAS1- 345 ACATTTTGGGCCCACAAAGT (SEQ ID NO: 989) Sp345 EPAS1- 346 GGGCCCACAAAGTGGGCCGT (SEQ ID NO: 990) Sp346 EPAS1- 347 GGCCCACAAAGTGGGCCGTC (SEQ ID NO: 991) Sp347 EPAS1- 348 GCCCACAAAGTGGGCCGTCG (SEQ ID NO: 992) Sp348 EPAS1- 349 TCCCCGACGGCCCACTTTGT (SEQ ID NO: 993) Sp349 EPAS1- 350 ATCCCCGACGGCCCACTTTG (SEQ ID NO: 994) Sp350 EPAS1- 351 CTCTGTGCGCTGATCCCCGA (SEQ ID NO: 995) Sp351 EPAS1- 352 GGATCAGCGCACAGAGTTCT (SEQ ID NO: 996) Sp352 EPAS1- 353 GATCAGCGCACAGAGTTCTT (SEQ ID NO: 997) Sp353 EPAS1- 354 GTTCTTGGGAGCAGCGCCGT (SEQ ID NO: 998) Sp354 EPAS1- 355 TTCTTGGGAGCAGCGCCGTT (SEQ ID NO: 999) Sp355 EPAS1- 356 TCTTGGGAGCAGCGCCGTTG (SEQ ID NO: 1000) Sp356 EPAS1- 357 GGAGAGACAGGGGGCCCCAA (SEQ ID NO: 1001) Sp357 EPAS1- 358 ACATGGGGTGGAGAGACAGG (SEQ ID NO: 1002) Sp358 EPAS1- 359 GACATGGGGTGGAGAGACAG (SEQ ID NO: 1003) Sp359 EPAS1- 360 AGACATGGGGTGGAGAGACA (SEQ ID NO: 1004) Sp360 EPAS1- 361 GAGACATGGGGTGGAGAGAC (SEQ ID NO: 1005) Sp361 EPAS1- 362 TTGAAGGTGGAGACATGGGG (SEQ ID NO: 1006) Sp362 EPAS1- 363 GTCTTGAAGGTGGAGACATG (SEQ ID NO: 1007) Sp363 EPAS1- 364 TGTCTTGAAGGTGGAGACAT (SEQ ID NO: 1008) Sp364 EPAS1- 365 TTGTCTTGAAGGTGGAGACA (SEQ ID NO: 1009) Sp365 EPAS1- 366 ATGTCTCCACCTTCAAGACA (SEQ ID NO: 1010) Sp366 EPAS1- 367 TGCCACTTACCTTGTCTTGA (SEQ ID NO: 1011) Sp367 EPAS1- 368 CGGGCTTGGCAGGTCTGCAA (SEQ ID NO: 1012) Sp368 EPAS1- 369 GGGCTTGGCAGGTCTGCAAA (SEQ ID NO: 1013) Sp369 EPAS1- 370 GGCAGGTCTGCAAAGGGTTT (SEQ ID NO: 1014) Sp370 EPAS1- 371 GCAGGTCTGCAAAGGGTTTT (SEQ ID NO: 1015) Sp371 EPAS1- 373 CAGGTCTGCAAAGGGTTTTG (SEQ ID NO: 1016) Sp372 EPAS1- 374 GCAAAGGGTTTTGGGGCTCG (SEQ ID NO: 1017) Sp373 EPAS1- 374 AGGCCCAGACGTGCTGAGTC (SEQ ID NO: 1018) Sp374 EPAS1- 375 TGGCCGGACTCAGCACGTCT (SEQ ID NO: 1019) Sp375 EPAS1- 376 ATGGCCGGACTCAGCACGTC (SEQ ID NO: 1020) Sp376 EPAS1- 377 AGACGTGCTGAGTCCGGCCA (SEQ ID NO: 1021) Sp377 EPAS1- 378 TTGGAGAGGGCTACCATGGC (SEQ ID NO: 1022) Sp378 EPAS1- 379 CTTGTTGGAGAGGGCTACCA (SEQ ID NO: 1023) Sp379 EPAS1- 380 CAGCTTCAGCTTGTTGGAGA (SEQ ID NO: 1024) Sp380 EPAS1- 381 TCAGCTTCAGCTTGTTGGAG (SEQ ID NO: 1025) Sp381 EPAS1- 382 TCGCTTCAGCTTCAGCTTGT (SEQ ID NO: 1026) Sp382 EPAS1- 383 GCTGAAGCTGAAGCGACAGC (SEQ ID NO: 1027) Sp383 EPAS1- 384 GTATGAAGAGCAAGCCTTCC (SEQ ID NO: 1028) Sp384 EPAS1- 385 CAAGCCTTCCAGGACCTGAG (SEQ ID NO: 1029) Sp385 EPAS1- 386 AAGCCTTCCAGGACCTGAGC (SEQ ID NO: 1030) Sp386 EPAS1- 387 AGCCTTCCAGGACCTGAGCG (SEQ ID NO: 1031) Sp387 EPAS1- 388 CACCCCGCTCAGGTCCTGGA (SEQ ID NO: 1032) Sp388 EPAS1- 389 GACTCACCCCGCTCAGGTCC (SEQ ID NO: 1033) Sp389 EPAS1- 390 GGGGATGACTCACCCCGCTC (SEQ ID NO: 1034) Sp390 EPAS1- 391 TACTCCCAGGGGGACCCACC (SEQ ID NO: 1035) Sp391 EPAS1- 392 TCCCAGGGGGACCCACCTGG (SEQ ID NO: 1036) Sp392 EPAS1- 393 GCCACCAGGTGGGTCCCCCT (SEQ ID NO: 1037) Sp393 EPAS1- 394 TGCCACCAGGTGGGTCCCCC (SEQ ID NO: 1038) Sp394 EPAS1- 395 GTGAGGTGCTGCCACCAGGT (SEQ ID NO: 1039) Sp395 EPAS1- 396 TGTGAGGTGCTGCCACCAGG (SEQ ID NO: 1040) Sp396 EPAS1- 397 AAATGTGAGGTGCTGCCACC (SEQ ID NO: 1041) Sp397 EPAS1- 398 GCAGCACCTCACATTTGATG (SEQ ID NO: 1042) Sp398 EPAS1- 399 CCTCACATTTGATGTGGAAA (SEQ ID NO: 1043) Sp399 EPAS1- 400 CCGTTTCCACATCAAATGTG (SEQ ID NO: 1044) Sp400 EPAS1- 401 GGAAACGGATGAAGAACCTC (SEQ ID NO: 1045) Sp401 EPAS1- 402 GAAACGGATGAAGAACCTCA (SEQ ID NO: 1046) Sp402 EPAS1- 403 AAACGGATGAAGAACCTCAG (SEQ ID NO: 1047) Sp403 EPAS1- 404 CGGATGAAGAACCTCAGGGG (SEQ ID NO: 1048) Sp404 EPAS1- 405 GGATGAAGAACCTCAGGGGT (SEQ ID NO: 1049) Sp405 EPAS1- 406 AAGGGCAGCTCCCACCCCTG (SEQ ID NO: 1050) Sp406 EPAS1- 407 TGGGAGCTGCCCTTTGATGC (SEQ ID NO: 1051) Sp407 EPAS1- 408 GTGGCTTGTCCGGCATCAAA (SEQ ID NO: 1052) Sp408 EPAS1- 409 AGTGGCTTGTCCGGCATCAA (SEQ ID NO: 1053) Sp409 EPAS1- 410 TTTGCGCTCAGTGGCTTGTC (SEQ ID NO: 1054) Sp410 EPAS1- 411 TTGGGTACATTTGCGCTCAG (SEQ ID NO: 1055) Sp411 EPAS1- 412 CTGAGCGCAAATGTACCCAA (SEQ ID NO: 1056) Sp412 EPAS1- 413 TGTGGCCGCTGCTCACCATT (SEQ ID NO: 1057) Sp413 EPAS1- 414 CTGTGGCCGCTGCTCACCAT (SEQ ID NO: 1058) Sp414 EPAS1- 415 AGTTCACCCAAAACCCCATG (SEQ ID NO: 1059) Sp415 EPAS1- 416 GTTCACCCAAAACCCCATGA (SEQ ID NO: 1060) Sp416 EPAS1- 417 TTCACCCAAAACCCCATGAG (SEQ ID NO: 1061) Sp417 EPAS1- 418 CAGGCCCCTCATGGGGTTTT (SEQ ID NO: 1062) Sp418 EPAS1- 419 CCAAAACCCCATGAGGGGCC (SEQ ID NO: 1063) Sp419 EPAS1- 420 CCAGGCCCCTCATGGGGTTT (SEQ ID NO: 1064) Sp420 EPAS1- 421 CAAAACCCCATGAGGGGCCT (SEQ ID NO: 1065) Sp421 EPAS1- 422 GATGGCCCAGGCCCCTCATG (SEQ ID NO: 1066) Sp422 EPAS1- 423 GGATGGCCCAGGCCCCTCAT (SEQ ID NO: 1067) Sp423 EPAS1- 424 GGGATGGCCCAGGCCCCTCA (SEQ ID NO: 1068) Sp424 EPAS1- 425 GATGTCTCAGGGGATGGCCC (SEQ ID NO: 1069) Sp425 EPAS1- 426 GCGGCAGATGTCTCAGGGGA (SEQ ID NO: 1070) Sp426 EPAS1- 427 GGCAGCGGCAGATGTCTCAG (SEQ ID NO: 1071) Sp427 EPAS1- 428 TGGCAGCGGCAGATGTCTCA (SEQ ID NO: 1072) Sp428 EPAS1- 429 GTGGCAGCGGCAGATGTCTC (SEQ ID NO: 1073) Sp429 EPAS1- 430 GCAGATGGAGGCTGTGGCAG (SEQ ID NO: 1074) Sp430 EPAS1- 431 CTGATGGCAGATGGAGGCTG (SEQ ID NO: 1075) Sp431 EPAS1- 432 CCTCCATCTGCCATCAGTCC (SEQ ID NO: 1076) Sp432 EPAS1- 433 CCGGGACTGATGGCAGATGG (SEQ ID NO: 1077) Sp433 EPAS1- 434 CTCCATCTGCCATCAGTCCC (SEQ ID NO: 1078) Sp434 EPAS1- 435 TCCATCTGCCATCAGTCCCG (SEQ ID NO: 1079) Sp435 EPAS1- 436 TCCCCGGGACTGATGGCAGA (SEQ ID NO: 1080) Sp436 EPAS1- 437 GCTGTTCTCCCCGGGACTGA (SEQ ID NO: 1081) Sp437 EPAS1- 438 CTGCTCTTGCTGTTCTCCCC (SEQ ID NO: 1082) Sp438 EPAS1- 439 CCGGGGAGAACAGCAAGAGC (SEQ ID NO: 1083) Sp439 EPAS1- 440 CCTGCTCTTGCTGTTCTCCC (SEQ ID NO: 1084) Sp440 EPAS1- 442 GGGTGGCGTAGCACTGTGGG (SEQ ID NO: 1085) Sp441 EPAS1- 442 TGGGTGGCGTAGCACTGTGG (SEQ ID NO: 1086) Sp442 EPAS1- 413 CTGGGTGGCGTAGCACTGTG (SEQ ID NO: 1087) Sp443 EPAS1- 444 ACTGGGTGGCGTAGCACTGT (SEQ ID NO: 1088) Sp444 EPAS1- 445 TACTGGGTGGCGTAGCACTG (SEQ ID NO: 1089) Sp445 EPAS1- 446 GTGCTACGCCACCCAGTACC (SEQ ID NO: 1090) Sp446 EPAS1- 447 GCTGTAGTCCTGGTACTGGG (SEQ ID NO: 1091) Sp447 EPAS1- 448 CAGGCTGTAGTCCTGGTACT (SEQ ID NO: 1092) Sp448 EPAS1- 449 ACAGGCTGTAGTCCTGGTAC (SEQ ID NO: 1093) Sp449 EPAS1- 450 CTGACGACAGGCTGTAGTCC (SEQ ID NO: 1094) Sp450 EPAS1- 451 CAGCCTGTCGTCAGCCCACA (SEQ ID NO: 1095) Sp451 EPAS1- 452 ACACCTTGTGGGCTGACGAC (SEQ ID NO: 1096) Sp452 EPAS1- 453 TCGTCAGCCCACAAGGTGTC (SEQ ID NO: 1097) Sp453 EPAS1- 454 TCAGCCCACAAGGTGTCAGG (SEQ ID NO: 1098) Sp454 EPAS1- 455 CAGCCCACAAGGTGTCAGGT (SEQ ID NO: 1099) Sp455 EPAS1- 456 ACACCTACCTGACACCTTGT (SEQ ID NO: 1100) Sp456 EPAS1- 457 CACACCCACCTGACACCTTG (SEQ ID NO: 1101) Sp457 EPAS1- 458 ACCAACCCTTCTTTCAGGCA (SEQ ID NO: 1102) Sp458 EPAS1- 459 TTCTTTCAGGCATGGCAAGC (SEQ ID NO: 1103) Sp459 EPAS1- 460 GGCATGGCAAGCCGGCTGCT (SEQ ID NO: 1104) Sp460 EPAS1- 461 GCATGGCAAGCCGGCTGCTC (SEQ ID NO: 1105) Sp461 EPAS1- 462 CAAATGAGGGCCCGAGCAGC (SEQ ID NO: 1106) Sp462 EPAS1- 463 AGCAGGTAGGACTCAAATGA (SEQ ID NO: 1107) Sp463 EPAS1- 464 CAGCAGGTAGGACTCAAATG (SEQ ID NO: 1108) Sp464 EPAS1- 465 GGTCAGTTCGGGCAGCAGGT (SEQ ID NO: 1109) Sp465 EPAS1- 466 ATCTGGTCAGTTCGGGCAGC (SEQ ID NO: 1110) Sp466 EPAS1- 467 CAGTCATATCTGGTCAGTTC (SEQ ID NO: 1111) Sp467 EPAS1- 468 ACAGTCATATCTGGTCAGTT (SEQ ID NO: 1112) Sp468 EPAS1- 469 ACTGACCAGATATGACTGTS (SEQ ID NO: 1113) Sp469 EPAS1- 470 GTTCACCTCACAGTCATATC (SEQ ID NO: 1114) Sp470 EPAS1- 471 TGAGGTGAACGTGCCCGTGC (SEQ ID NO: 1115) Sp471 EPAS1- 472 GAGGTGAACGTGCCCGTGCT (SEQ ID NO: 1116) Sp472 EPAS1- 473 AGCGTGGAGCTTCCCAGCAC (SEQ ID NO: 1117) Sp473 EPAS1- 474 GAGCGTGGAGCTTCCCAGCA (SEQ ID NO: 1118) Sp474 EPAS1- 475 GGAAGCTCCACGCTCCTGCA (SEQ ID NO: 1119) Sp475 EPAS1- 476 AGCTCCACGCTCCTGCAAGG (SEQ ID NO: 1120) Sp476 EPAS1- 477 GCTCCACGCTCCTGCAAGGA (SEQ ID NO: 1121) Sp477 EPAS1- 478 CTCCACGCTCCTGCAAGGAG (SEQ ID NO: 1122) Sp478 EPAS1- 479 GTCCCCTCCTTGCAGGAGCG (SEQ ID NO: 1123) Sp479 EPAS1- 480 TGAGGAGGTCCCCTCCTTGC (SEQ ID NO: 1124) Sp480 EPAS1- 481 AGGGGACCTCCTCAGAGCCC (SEQ ID NO: 1125) Sp481 EPAS1- 482 CCTCCTCAGAGCCCTGGACC (SEQ ID NO: 1126) Sp482 EPaS1- 483 CCTGGTCCAGGGCTCTGAGG (SEQ ID NO: 1127) Sp483 EPAS1- 484 TGGCCTGGTCCAGGGCTCTG (SEQ ID NO: 1128) Sp484 EPAS1- 485 GGCTCAGGTGGCCTGGTCCA (SEQ ID NO: 1129) Sp485 EPAS1- 486 TGGCTCAGGTGGCCTGGTCC (SEQ ID NO: 1130) Sp486 EPAS1- 487 TGGACCAGGCCACCTGAGCC (SEQ ID NO: 1131) Sp487 EPAS1- 488 AAGGCCTGGCTCAGGTGGCC (SEQ ID NO: 1132) Sp488 EPAS1- 489 GGTAGAAGGCCTGGCTCAGG (SEQ ID NO: 1133) Sp489

TABLE 13 Target sequences of ANGPT2 gene for SpCas9 Gene No. Target sequence ANGPT2- 1 TCTGAGCTGTGATCTTGTCT (SEQ ID NO: 1134) Sp1 ANGPT2- 2 CCGCAGCCTATAACAACTTT (SEQ ID NO: 1135) Sp2 ANGPT2- 3 CCGAAAGTTGTTATAGGCTG (SEQ ID NO: 1136) Sp3 ANGPT2- 4 GCTCTTCCGAAAGTTGTTAT (SEQ ID NO: 1137) Sp4 ANGPT2- 5 TAACAACTTTCGGAAGAGCA (SEQ ID NO: 1138) Sp5 ANGPT2- 6 CGGAAGAGCATGGACAGCAT (SEQ ID NO: 1139) Sp6 ANGPT2- 7 CATAGGAAAGAAGCAATATC (SEQ ID NO: 1140) Sp7 ANGPT2- 8 AAGCAATATCAGGTCCAGCA (SEQ ID NO: 1141) Sp8 ANGPT2- 9 AGCAATATCAGGTCCAGCAT (SEQ ID NO: 1142) Sp9 ANGPT2- 10 TGTAGCTGCAGGACCCATGC (SEQ ID NO: 1143) Sp10 ANGPT2- 11 CAGGAGGAAAGTGTAGCTGC (SEQ ID NO: 1144) Sp11 ANGPT2- 12 CACTTTCCTCCTGCCAGAGA (SEQ ID NO: 1145) Sp12 ANGPT2- 13 AGTTGTCCATCTCTGGCAGG (SEQ ID NO: 1146) Sp13 ANGPT2- 14 GGCAGTTGTCCATCTCTGGC (SEQ ID NO: 1147) Sp14 ANGPT2- 15 GAGCGGCAGTTGTCCATCTC (SEQ ID NO: 1148) Sp15 ANGPT2- 16 CGTAGGGGCTGGAGGAAGAG (SEQ ID NO: 1149) Sp16 ANGPT2- 17 ATTGGACACGTAGGGGCTGG (SEQ ID NO: 1150) Sp17 ANGPT2- 18 AGCATTGGACACGTAGGGGC (SEQ ID NO: 1151) Sp18 ANGPT2- 19 GCACAGCATTGGACACGTAG (SEQ ID NO: 1152) Sp19 ANGPT2- 20 TGCACAGCATTGGACACGTA (SEQ ID NO: 1153) Sp20 ANGPT2- 21 CTGCACAGCATTGGACACGT (SEQ ID NO: 1154) Sp21 ANGPT2- 22 ACGTGTCCAATGCTGTGCAG (SEQ ID NO: 1155) Sp22 ANGPT2- 23 CGTGTCCAATGCTGTGCAGA (SEQ ID NO: 1156) Sp23 ANGPT2- 24 CGCGTCCCTCTGCACAGCAT (SEQ ID NO: 1157) Sp24 ANGPT2- 25 GCCGCTCGAATACGATGACT (SEQ ID NO: 1158) Sp25 ANGPT2- 26 ACCGAGTCATCGTATTCGAG (SEQ ID NO: 1159) Sp26 ANGPT2- 27 AATACGATGACTCGGTGCAG (SEQ ID NO: 1160) Sp27 ANGPT2- 28 GGTGCAGAGGCTGCAAGTGC (SEQ ID NO: 1161) Sp28 ANGPT2- 29 GCAAGTGCTGGAGAACATCA (SEQ ID NO: 1162) Sp29 ANGPT2- 30 TCATGGAAAACAACACTCAG (SEQ ID NO: 1163) Sp30 ANGPT2- 31 CAACACTCAGTGGCTAATGA (SEQ ID NO: 1164) Sp31 ANGPT2- 32 ACTCAGTGGCTAATGAAGGT (SEQ ID NO: 1165) Sp32 ANGPT2- 33 CTAGCTTGAGAATTATATCC (SEQ ID NO: 1166) Sp33 ANGPT2- 34 TTTCTTTCTTCATGTTGTCC (SEQ ID NO: 1167) Sp34 ANGPT2- 35 GGACAACATGAAGAAAGAAA (SEQ ID NO: 1168) Sp35 ANGPT2- 36 GAATGCAGTACAGAACCAGA (SEQ ID NO: 1169) Sp36 ANGPT2- 37 TTTCTATCATCACAGCCGTC (SEQ ID NO: 1170) Sp37 ANGPT2- 38 ACGGCTGTGATGATAGAAAT (SEQ ID NO: 1171) Sp38 ANGPT2- 39 CGGCTGTGATGATAGAAATA (SEQ ID NO: 1172) Sp39 ANGPT2- 40 AAACCTGTTGAACCAAACAG (SEQ ID NO: 1173) Sp40 ANGPT2- 41 GCTCCGCTGTTTGGTTCAAC (SEQ ID NO: 1174) Sp41 ANGPT2- 42 ACCAAACAGCGGAGCAAACG (SEQ ID NO: 1175) Sp42 ANGPT2- 43 TCCGCGTTTGCTCCGCTGTT (SEQ ID NO: 1176) Sp43 ANGPT2- 44 AACGCGGAAGTTAACTGATG (SEQ ID NO: 1177) Sp44 ANGPT2- 45 CTAGCTTGAGAATTATATCC (SEQ ID NO: 1178) Sp45 ANGPT2- 46 TTTCTTTCTTCATGTTGTCC (SEQ ID NO: 1179) Sp46 ANGPT2- 47 GGACAACATGAAGAAAGAAA (SEQ ID NO: 1180) Sp47 ANGPT2- 48 GAATGCAGTACAGAACCAGA (SEQ ID NO: 1181) Sp48 ANGPT2- 49 TTTCTATCATCACAGCCGTC (SEQ ID NO: 1182) Sp49 ANGPT2- 50 ACGGCTGTGATGATAGAAAT (SEQ ID NO: 1183) Sp50 ANGPT2- 51 CGGCTGTGATGATAGAAATA (SEQ ID NO: 1184) Sp51 ANGPT2- 52 AAACCTGTTGAACCAAACAG (SEQ ID NO: 1185) Sp52 ANGPT2- 53 GCTCCGCTGTTTGGTTCAAC (SEQ ID NO: 1186) Sp53 ANGPT2- 54 ACCAAACAGCGGAGCAAACG (SEQ ID NO: 1187) Sp54 ANGPT2- 55 TCCGCGTTTGCTCCGCTGTT (SEQ ID NO: 1188) Sp55 ANGPT2- 56 AACGCGGAAGTTAACTGATG (SEQ ID NO: 1189) Sp56 ANGPT2- 57 TACAAGTTTCCTAGAAAAGA (SEQ ID NO: 1190) Sp57 ANGPT2- 58 TAGCTAGCACCTTCTTTTCT (SEQ ID NO: 1191) Sp58 ANGPT2- 59 AGAAAAGAAGGTGCTAGCTA (SEQ ID NO: 1192) Sp59 ANGPT2- 60 CTTCTTTTTATTGACTGTAGT (SEQ ID NO: 1193) Sp60 ANGPT2- 61 AGAAGAGAAAGATCAGCTAC (SEQ ID NO: 1194) Sp61 ANGPT2- 62 TTCAATGATGGAATTTTGCT (SEQ ID NO: 1195) Sp62 ANGPT2- 63 TTTTTCTAGTTCTTTCAATGA (SEQ ID NO: 1196) Sp63 ANGPT2- 64 AAAAAAAATAGTGACTGCCA (SEQ ID NO: 1197) Sp64 ANGPT2- 65 AAGAACTGAATTATTCACCG (SEQ ID NO: 1198) Sp65 ANGPT2- 66 GAAGCAGCAACATGATCTCA (SEQ ID NO: 1199) Sp66 ANGPT2- 67 TGTAAACTTACAGTTTGATG (SEQ ID NO: 1200) Sp67 ANGPT2- 68 CTATTTTTTAAAAGCAGCTA (SEQ ID NO: 1201) Sp68 ANGPT2- 69 GTTCTTCTTTAGCAACAGTG (SEQ ID NO: 1202) Sp69 ANGPT2- 70 TGTTCTTCTTTAGCAACAGT (SEQ ID NO: 1203) Sp70 ANGPT2- 71 TTGTTCTTCTTTAGCAACAG (SEQ ID NO: 1204) Sp71 ANGPT2- 72 TGTGCTGAAGTATTCAAATC (SEQ ID NO: 1205) Sp72 ANGPT2- 73 AAATCAGGACACACCACGAA (SEQ ID NO: 1206) Sp73 ANGPT2- 74 TAACGTGTAGATGCCATTCG (SEQ ID NO: 1207) Sp74 ANGPT2- 75 TGATCTCTTCTGTAGAATTA (SEQ ID NO: 1208) Sp75 ANGPT2- 76 TTGATCTCTTCTGTAGAATT (SEQ ID NO: 1209) Sp76 ANGPT2- 77 TAATTCTACAGAAGAGATCA (SEQ ID NO: 1210) Sp77 ANGPT2- 78 CTACAGAAGAGATCAAGGTG (SEQ ID NO: 1211) Sp78 ANGPT2- 79 TTTGCAGGCCTACTGTGACA (SEQ ID NO: 1212) Sp79 ANGPT2- 80 GCCTACTGTGACATGGAAGC (SEQ ID NO: 1213) Sp80 ANGPT2- 81 TCCAGCTTCCATGTCACAGT (SEQ ID NO: 1214) Sp81 ANGPT2- 82 TACTGTGACATGGAAGCTGG (SEQ ID NO: 1215) Sp82 ANGPT2- 83 TGTGACATGGAAGCTGGAGG (SEQ ID NO: 1216) Sp83 ANGPT2- 84 GACATGGAAGCTGGAGGAGG (SEQ ID NO: 1217) Sp84 ANGPT2- 85 ACATGGAAGCTGGAGGAGGC (SEQ ID NO: 1218) Sp85 ANGPT2- 86 TGGAAGCTGGAGGAGGCGGG (SEQ ID NO: 1219) Sp86 ANGPT2- 87 GACAATTATTCAGCGACGTG (SEQ ID NO: 1220) Sp87 ANGPT2- 88 ATTATTCAGCGACGTGAGGA (SEQ ID NO: 1221) Sp88 ANGPT2- 89 ATGGCAGCGTTGATTTTCAG (SEQ ID NO: 1222) Sp89 ANGPT2- 90 GCGTTGATTTTCAGAGGACT (SEQ ID NO: 1223) Sp90 ANGPT2- 91 GACTTGGAAAGAATATAAAG (SEQ ID NO: 1224) Sp91 ANGPT2- 92 GGAAAGAATATAAAGTGGTA (SEQ ID NO: 1225) Sp92 ANGPT2- 93 CAGGGATTTGGTAACCCTTC (SEQ ID NO: 1226) Sp93 ANGPT2- 94 GTAACCCTTCAGGAGAATAT (SEQ ID NO: 1227) Sp94 ANGPT2- 95 CCCTTCAGGAGAATATTGGC (SEQ ID NO: 1228) Sp95 ANGPT2- 96 CCAGCCAATATTCTCCTGAA (SEQ ID NO: 1229) Sp56 ANGPT2- 97 CCTTCAGGAGAATATTGGCT (SEQ ID NO: 1230) Sp97 ANGPT2- 98 CCCAGCCAATATTCTCCTGA (SEQ ID NO: 1231) Sp98 ANGPT2- 99 TTAAAATACACCTTAAAGAC (SEQ ID NO: 1232) Sp99 ANGPT2- 100 TAAAATACACCTTAAAGACT (SEQ ID NO: 1233) Sp100 ANGPT2- 101 ATACACCTTAAAGACTGGGA (SEQ ID NO: 1234) Sp101 ANGPT2- 102 TACACCTTAAAGACTGGGAA (SEQ ID NO: 1235) Sp102 ANGPT2- 103 CATTCCCTTCCCAGTCTTTA (SEQ ID NO: 1236) Sp103 ANGPT2- 104 TAAAGACTGGGAAGGGAATG (SEQ ID NO: 1237) Sp104 ANGP12- 105 CAAGTGAAGAACTCAATTAT (SEQ ID NO: 1238) Sp105 ANGPT2- 106 GCTTACAGGATTCACCTTAA (SEQ ID NO: 1239) Sp106 ANGPT2- 107 ATTCACCTTAAAGGACTTAC (SEQ ID NO: 1240) Sp107 ANGPT2- 108 TTCACCTTAAAGGACTTACA (SEQ ID NO: 1241) Sp108 ANGPT2- 109 CTGTCCCTGTAAGTCCTTTA (SEQ ID NO: 1242) Sp109 ANGPT2- 110 AAAGGACTTACAGGGACAGC (SEQ ID NO: 1243) Sp110 ANGPT2- 111 GCTGATGCTGCTTATTTTGC (SEQ ID NO: 1244) Sp111 ANGPT2- 112 ATAAGCAGCATCAGCCAACC (SEQ ID NO: 1245) Sp112 ANGPT2- 113 TGCTAAAATCATTTCCTGGT (SEQ ID NO: 1246) Sp113 ANGPT2- 114 TTTGTGCTAAAATCATTTCC (SEQ ID NO: 1247) Sp114 ANGPT2- 115 AGGAAATGATTTTAGCACAA (SEQ ID NO: 1248) Sp115 ANGPT2- 116 AATGATTTTAGCACAAAGGA (SEQ ID NO: 1249) Sp116 ANGPT2- 117 AAATGTTCACAAATGCTAAC (SEQ ID NO: 1250) Sp117 ANGPT2- 118 TGTTCACAAATGCTAACAGG (SEQ ID NO: 1251) Sp118 ANGPT2- 119 CACAAATGCTAACAGGAGGT (SEQ ID NO: 1252) Sp119 ANGPT2- 120 ACAAATGCTAACAGGAGGTA (SEQ ID NO: 1253) Sp120 ANGPT2- 121 GGCTGGTGGTTTGATGCATG (SEQ ID NO: 1254) Sp121 ANGPT2- 122 TGTGGTCCTTCCAACTTGAA (SEQ ID NO: 1255) Sp122 ANGPT2- 123 TACATTCCGTTCAAGTTTGGA (SEQ ID NO: 1256) Sp123 ANGPT2- 124 ATAGTACATTCCGTTCAAGT (SEQ ID NO: 1257) Sp124 ANGPT2- 125 ACGGAATGTACTATCCACAG (SEQ ID NO: 1258) Sp125 ANGPT2- 126 TTATTTGTGTTCTGCCTCTG (SEQ ID NO: 1259) Sp126 ANGPT2- 127 CAGAACACAAATAAGTTCAA (SEQ ID NO: 1260) Sp127 ANGPT2- 128 ATAAGTTCAACGGCATTAAA (SEQ ID NO: 1261) Sp128 ANGPT2- 129 ACGGCATTAAATGGTACTAC (SEQ ID NO: 1262) Sp129 ANGPT2- 130 ATTAAATGGTACTACTGGAA (SEQ ID NO: 1263) Sp130 ANGPT2- 131 TGGTACTACTGGAAAGGCTC (SEQ ID NO: 1264) Sp131 ANGPT2- 132 AGGCTCAGGCTATTCGCTCA (SEQ ID NO: 1265) Sp132 ANGPT2- 133 TGGTCGGATCATCATGGTTG (SEQ ID NO: 1266) Sp133 ANGPT2- 134 ATCTGCTGGTCGGATCATCA (SEQ ID NO: 1267) Sp134 ANGPT2- 135 ATGTTTAGAAATCTGCTGGT (SEQ ID NO: 1268) Sp135 ANGPT2- 136 TGGGATGTTTAGAAATCTGC (SEQ ID NO: 1269) Sp136

TABLE 14 Target sequences of ANGPTL4 gene for SpCas9 Gene No. Target sequence ANGPTL4-Sp1 1 AGGCTACCTAAGAGGATGAG (SEQ ID NO: 1270) ANGPTL4-Sp2 2 GAGGATGAGCGGTGCTCCGA (SEQ ID NO: 1271) ANGPTL4-Sp3 3 ATGAGCGGTGCTCCGACGGC (SEQ ID NO: 1272) ANGPTL4-Sp4 4 TGAGCGGTGCTCCGACGGCC (SEQ ID NO: 1273) ANGPTL4-Sp5 5 GAGCGGTGCTCCGACGGCCG (SEQ ID NO: 1274) ANGPTL4-Sp6 6 ATCAGGGCTGCCCCGGCCGT (SEQ ID NO: 1275) ANGPTL4-Sp7 7 GCAGAGCATCAGGGCTGCCC (SEQ ID NO: 1276) ANGPTL4-Sp8 8 GGTGGCGGCGCAGAGCATCA (SEQ ID NO: 1277) ANGPTL4-Sp9 9 CGGTGGCGGCGCAGAGCATC (SEQ ID NO: 1278) ANGPTL4-Sp10 10 GCTCAGTAGCACGGCGGTGG (SEQ ID NO: 1279) ANGPTL4-Sp11 11 AGCGCTCAGTAGCACGGCGG (SEQ ID NO: 1280) ANGPTL4-Sp12 12 CTGAGCGCTCAGTAGCACGG (SEQ ID NO: 1281) ANGPTL4-Sp13 13 CGCCGTGCTACTGAGCGCTC (SEQ ID NO: 1282) ANGPTL4-Sp14 14 GCCGTGCTACTGAGCGCTCA (SEQ ID NO: 1283) ANGPTL4-Sp15 15 GCCCTGAGCGCTCAGTAGCA (SEQ ID NO: 1284) ANGPTL4-Sp16 16 GTGCTACTGAGCGCTCAGGG (SEQ ID NO: 1285) ANGPTL4-Sp17 17 CGCGGCGACTTGGACTGCAC (SEQ ID NO: 1286) ANGPTL4-Sp18 18 GCGCGGCGACTTGGACTGCA (SEQ ID NO: 1287) ANGPTL4-Sp19 19 GGACGCAAAGCGCGGCGACT (SEQ ID NO: 1288) ANGPTL4-Sp20 20 AGTCGCCGCGCTTTGCGTCC (SEQ ID NO: 1289) ANGPTL4-Sp21 21 GTCGCCGCGCTTTGCGTCCT (SEQ ID NO: 1290) ANGPTL4-Sp22 22 TCGTCCCAGGACGCAAAGCG (SEQ ID NO: 1291) ANGPTL4-Sp23 23 CAGGACATTCATCTCGTCCC (SEQ ID NO: 1292) ANGPTL4-Sp24 24 CTGGGACGAGATGAATGTCC (SEQ ID NO: 1293) ANGPTL4-Sp25 25 GAGATGAATGTCCTGGCGCA (SEQ ID NO: 1294) ANGPTL4-Sp26 26 GCTGCAGGAGTCCGTGCGCC (SEQ ID NO: 1295) ANGPTL4-Sp27 27 GCGCACGGACTCCTGCAGCT (SEQ ID NO: 1296) ANGPTL4-Sp28 28 CGGACTCCTGCAGCTCGGCC (SEQ ID NO: 1297) ANGPTL4-Sp28 29 GGACTCCTGCAGCTCGGCCA (SEQ ID NO: 1298) ANGPTL4-Sp30 30 GACTCCTGCAGCTCGGCCAG (SEQ ID NO: 1299) ANGPTL4-Sp31 31 GCAGCCCCTGGCCGAGCTGC (SEQ ID NO: 1300) ANGPTL4-Sp32 32 CCAGGGGCTGCGCGAACACG (SEQ ID NO: 1301) ANGPTL4-Sp33 33 CCGCGTGTTCGCGCAGCCCC (SEQ ID NO: 1302) ANGPTL4-Sp34 34 CAGCGCGCTCAGCTGACTGC (SEQ ID NO: 1303) ANGPTL4-Sp35 35 CCGCAGTCAGCTGAGCGCGC (SEQ ID NO: 1304) ANGPTL4-Sp36 36 CCAGCGCGCTCAGCTGACTG (SEQ ID NO: 1305) ANGPTL4-Sp37 37 GTCAGCTGAGCGCGCTGGAG (SEQ ID NO: 1306) ANGPTL4-Sp38 38 GAGCGGCGCCTGAGCGCGTG (SEQ ID NO: 1307) ANGPTL4-Sp39 39 AGCGGCGCCTGAGCGCGTGC (SEQ ID NO: 1308) ANGPTL4-Sp40 40 AGGCGGACCCGCACGCGCTC (SEQ ID NO: 1309) ANGPTL4-Sp41 41 CGCGTGCGGGTCCGCCTGTC (SEQ ID NO: 1310) ANGPTL4-Sp42 42 GCGTGCGGGTCCGCCTGTCA (SEQ ID NO: 1311) ANGPTL4-Sp43 43 GTCCGCCTGTCAGGGAACCG (SEQ ID NO: 1312) ANGPTL4-Sp44 44 TCCGCCTGTCAGGGAACCGA (SEQ ID NO: 1313) ANGPTL4-Sp45 45 CCGCCTGTCAGGGAACCGAG (SEQ ID NO: 1314) ANGPTL4-Sp46 46 CCCCTCGGTTCCCTGACAGG (SEQ ID NO: 1315) ANGPTL4-Sp47 47 GGACCCCTCGGTTCCCTGAC (SEQ ID NO: 1316) ANGPTL4-Sp48 48 CGGGAGGTCGGTGGACCCCT (SEQ ID NO: 1317) ANGPTL4-Sp49 49 AGGGGCTAACGGGAGGTCGG (SEQ ID NO: 1318) ANGPTL4-Sp50 50 CTCAGGGGCTAACGGGAGGT (SEQ ID NO: 1319) ANGPTL4-Sp51 51 GGCTCTCAGGGGCTAACGGG (SEQ ID NO: 1320) ANGPTL4-Sp52 52 TCCCGTTAGCCCCTGAGAGC (SEQ ID NO: 1321) ANGPTL4-Sp53 53 CCCGTTAGCCCCTGAGAGCC (SEQ ID NO: 1322) ANGPTL4-Sp54 54 CCCGGCTCTCAGGGGCTAAC (SEQ ID NO: 1323) ANGPTL4-Sp55 55 ACCCGGCTCTCAGGGGCTAA (SEQ ID NO: 1324) ANGPTL4-Sp56 56 GTTAGCCCCTGAGAGCCGGG (SEQ ID NO: 1325) ANGPTL4-Sp57 57 AGGGTCCACCCGGCTCTCAG (SEQ ID NO: 1326) ANGPTL4-Sp58 58 CAGGGTCCACCCGGCTCTCA (SEQ ID NO: 1327) ANGPTL4-Sp59 59 TCAGGGTCCACCCGGCTCTC (SEQ ID NO: 1328) ANGPTL4-Sp60 60 TGAGAGCCGGGTGGACCCTG (SEQ ID NO: 1329) ANGPTL4-Sp61 61 GAAGGACCTCAGGGTCCACC (SEQ ID NO: 1330) ANGPTL4-Sp62 62 GCAGGCTGTGAAGGACCTCA (SEQ ID NO: 1331) ANGPTL4-Sp63 63 TGCAGGCTGTGAAGGACCTC (SEQ ID NO: 1332) ANGPTL4-Sp64 64 TGAGGTCCTTCACAGCCTGC (SEQ ID NO: 1333) ANGPTL4-Sp65 65 CACGTACCTGCAGGCTGTGA (SEQ ID NO: 1334) ANGPTL4-Sp66 66 CCCTGGGGACACGTACCTGC (SEQ ID NO: 1335) ANGPTL4-Sp67 67 CCCTCCCCAGACACAACTCA (SEQ ID NO: 1336) ANGPTL4-Sp68 68 TGAGCCTTGAGTTGTGTCTG (SEQ ID NO: 1337) ANGPTL4-Sp69 69 CTGAGCCTTGAGTTGTGTCT (SEQ ID NO: 1338) ANGPTL4-Sp70 70 TCTGAGCCTTGAGTTGTGTC (SEQ ID NO: 1339) ANGPTL4-Sp71 71 AACTCAAGGCTCAGAACAGC (SEQ ID NO: 1340) ANGPTL4-Sp72 72 GATCCAGCAACTCTTCCACA (SEQ ID NO: 1341) ANGPTL4-Sp73 73 CCAGCAACTCTTCCACAAGG (SEQ ID NO: 1342) ANGPTL4-Sp74 74 CCACCTTGTGGAAGAGTTGC (SEQ ID NO: 1343) ANGPTL4-Sp75 75 GCTGCTGCTGGGCCACCTTG (SEQ ID NO: 1344) ANGPTL4-Sp76 76 ACAAGGTGGCCCAGCAGCAG (SEQ ID NO: 1345) ANGPTL4-Sp77 77 GGCCCAGCAGCAGCGGCACC ANGPTL4-Sp78 78 CTCCAGGTGCCGCTGCTGCT (SEQ ID NO: 1347) ANGPTL4-Sp79 79 TCTCCAGGTGCCGCTGCTGC (SEQ ID NO: 1348) ANGPTL4-Sp80 80 TTCGCAGGTGCTGCTTCTCC (SEQ ID NO: 1349) ANGPTL4-Sp81 81 TTTGCAGATGCTGAATTCGC (SEQ ID NO: 1350) ANGPTL4-Sp82 82 AATTCAGCATCTGCAAAGCC (SEQ ID NO: 1351) ANGPTL4-Sp83 83 CCCTTGATCCTAGGGTTACC (SEQ ID NO: 1352) ANGPTL4-Sp84 84 CCCATCCTAGTTTGGCCTCC (SEQ ID NO: 1353) ANGPTL4-Sp85 85 GTGGTCCAGGAGGCCAAACT (SEQ ID NO: 1354) ANGPTL4-Sp86 86 CTAGGTGCTTGTGGTCCAGG (SEQ ID NO: 1355) ANGPTL4-Sp87 87 GGTCTAGGTGCTTGTGGTCC (SEQ ID NO: 1356) ANGPTL4-Sp88 88 CCACAAGCACCTAGACCATG (SEQ ID NO: 1357) ANGPTL4-Sp89 89 CCTCATGGTCTAGGTGCTTG (SEQ ID NO: 1358) ANGPTL4-Sp90 90 CAAGCACCTAGACCATGAGG (SEQ ID NO: 1359) ANGPTL4-Sp91 91 GCTTGGCCACCTCATGGTCT (SEQ ID NO: 1360) ANGPTL4-Sp92 92 GGGCAGGCTTGGCCACCTCA (SEQ ID NO: 1361) ANGPTL4-Sp93 93 CCAAGCCTGCCCGAAGAAAG (SEQ ID NO: 1362) ANGPTL4-Sp94 94 CCTCTTTCTTCGGGCAGGCT (SEQ ID NO: 1363) ANGPTL4-Sp95 95 GGCAGCCTCTTTCTTCGGGC (SEQ ID NO: 1364) ANGPTL4-Sp96 96 CTCGGGCAGCCTCTTTCTTC (SEQ ID NO: 1365) ANGPTL4-Sp97 97 TCTCGGGCAGCCTCTTTCTT (SEQ ID NO: 1366) ANGPTL4-Sp98 98 AAGAAAGAGGCTGCCCGAGA (SEQ ID NO: 1367) ANGPTL4-Sp99 99 TCAACTGGCTGGGCCATCTC (SEQ ID NO: 1368) ANGPTL4-Sp100 100 GTCAACTGGCTGGGCCATCT (SEQ ID NO: 1369) ANGPTL4-Sp101 101 GATGGCCCAGCCAGTTGACC (SEQ ID NO: 1370) ANGPTL4-Sp102 102 GTGAGCCGGGTCAACTGGCT (SEQ ID NO: 1371) ANGPTL4-Sp103 103 TGTGAGCCGGGTCAACTGGC (SEQ ID NO: 1372) ANGPTL4-Sp104 104 ACATTGTGAGCCGGGTCAAC (SEQ ID NO: 1373) ANGPTL4-Sp105 105 GGCGGCTGACATTGTGAGCC (SEQ ID NO: 1374) ANGPTL4-Sp106 106 AGGCGGCTGACATTGTGAGC (SEQ ID NO: 1375) ANGPTL4-Sp107 107 GCAGACACTCACGGTGCAGG (SEQ ID NO: 1376) ANGPTL4-Sp108 108 GGGGCAGACACTCACGGTGC (SEQ ID NO: 1377) ANGPTL4-Sp109 109 ATCTCCCTTCAGGGCTGCCC (SEQ ID NO: 1378) ANGPTL4-Sp110 110 TCTCCCTTCAGGGCTGCCCA (SEQ ID NO: 1379) ANGPTL4-Sp111 111 AGGGCTGCCCAGGGATTGCC (SEQ ID NO: 1380) ANGPTL4-Sp112 112 AACAGCTCCTGGCAATCCCT (SEQ ID NO: 1381) ANGPTL4-Sp113 113 GAACAGCTCCTGGCAATCCC (SEQ ID NO: 1382) ANGPTL4-Sp114 114 GGATTGCCAGGAGCTGTTCC (SEQ ID NO: 1383) ANGPTL4-Sp115 115 TGCCAGGAGCTGTTCCAGGT (SEQ ID NO: 1384) ANGPTL4-Sp116 116 CCAGGAGCTGTTCCAGGTTG (SEQ ID NO: 1385) ANGPTL4-Sp117 117 CCCCAACCTGGAACAGCTCC (SEQ ID NO: 1386) ANGPTL4-Sp118 118 AGCTGTTCCAGGTTGGGGAG (SEQ ID NO: 1387) ANGPTL4-Sp119 119 CACTCTGCCTCTCCCCAACC (SEQ ID NO: 1388) ANGPTL4-Sp120 120 CAGGTTGGGGAGAGGCACAG (SEQ ID NO: 1389) ANGPTL4-Sp121 121 ACTATTTGAAATCCAGCCTC (SEQ ID NO: 1390) ANGPTL4-Sp122 122 CTATTTGAAATCCAGCCTCA (SEQ ID NO: 1391) ANGPTL4-Sp123 123 TATTTGAAATCCAGCCTCAG (SEQ ID NO: 1392) ANGPTL4-Sp124 124 ATGGCGGAGACCCCTGAGGC (SEQ ID NO: 1393) ANGPTL4-Sp125 125 AAAAATGGCGGAGACCCCTG (SEQ ID NO: 1394) ANGPTL4-Sp126 126 TCAGGGGTCTCCGCCATTTT (SEQ ID NO: 1395) ANGPTL4-Sp127 127 TTGCAGTTCACCAAAAATGG (SEQ ID NO: 1396) ANGPTL4-Sp128 128 ATCTTGCAGTTCACCAAAAA (SEQ ID NO: 1397) ANGPTL4-Sp129 129 GTGAACTGCAAGATGACCTC (SEQ ID NO: 1398) ANGPTL4-Sp130 130 ACTGCAAGATGACCTCAGGT (SEQ ID NO: 1399) ANGPTL4-Sp131 131 CTGCAAGATGACCTCAGGTA (SEQ ID NO: 1400) ANGPTL4-Sp132 132 GGACTAACACACCCTACCTG (SEQ ID NO: 1401) ANGPTL4-Sp133 133 GTACCTTTCTGGGCAGATGG (SEQ ID NO: 1402) ANGPTL4-Sp134 134 CTTTCTGGGCAGATGGAGGC (SEQ ID NO: 1403) ANGPTL4-Sp135 135 GAGGCTGGACAGTAATTCAG (SEQ ID NO: 1404) ANGPTL4-Sp136 136 GTAATTCAGAGGCGCCACGA (SEQ ID NO: 1405) ANGPTL4-Sp137 137 GAGGCGCCACGATGGCTCAG (SEQ ID NO: 1406) ANGPTL4-Sp138 138 TGAAGTCCACTGAGCCATCG (SEQ ID NO: 1407) ANGPTL4-Sp139 139 ATGGCTCAGTGGACTTCAAC (SEQ ID NO: 1408) ANGPTL4-Sp140 140 CAGTGGACTTCAACCGGCCC (SEQ ID NO: 1409) ANGPTL4-Sp141 141 AGTGGACTTCAACCGGCCCT (SEQ ID NO: 1410) ANGPTL4-Sp142 142 CCGGCCCTGGGAAGCCTACA (SEQ ID NO: 1411) ANGPTL4-Sp143 143 CCTTGTAGGCTTCCCAGGGC (SEQ ID NO: 1412) ANGPTL4-Sp144 144 GCCCTGGGAAGCCTACAAGG (SEQ ID NO: 1413) ANGPTL4-Sp145 145 CCCTGGGAAGCCTACAAGGC (SEQ ID NO: 1414) ANGPTL4-Sp146 146 CCCGCCTTGTAGGCTTCCCA (SEQ ID NO: 1415) ANGPTL4-Sp147 147 CCTGGGAAGCCTACAAGGCG (SEQ ID NO: 1416) ANGPTL4-Sp148 148 CCCCGCCTTGTAGGCTTCCC (SEQ ID NO: 1417) ANGPTL4-Sp149 149 GAAGCCTACAAGGCGGGGTT (SEQ ID NO: 1418) ANGPTL4-Sp150 150 AAGCCTACAAGGCGGGGTTT (SEQ ID NO: 1419) ANGPTL4-Sp151 151 AGCCTACAAGGCGGGGTTTG (SEQ ID NO: 1420) ANGPTL4-Sp152 152 ATCCCCAAACCCCGCCTTGT (SEQ ID NO: 1421) ANGPTL4-Sp153 153 GCGGGGTTTGGGGATCCCCA (SEQ ID NO: 1422) ANGPTL4-Sp154 154 GGTTTGGGGATCCCCACGGT (SEQ ID NO: 1423) ANGPTL4-Sp155 155 CACTAGAAACACCTACCGTG (SEQ ID NO: 1424) ANGPTL4-Sp156 156 CCACTAGAAACACCTACCGT (SEQ ID NO: 1425) ANGPTL4-Sp157 157 CTCCCACTCCAGGCGAGTTC (SEQ ID NO: 1426) ANGPTL4-Sp158 158 CACTCCAGGCGAGTTCTGGC (SEQ ID NO: 1427) ANGPTL4-Sp159 159 ACTCCAGGCGAGTTCTGGCT (SEQ ID NO: 1428) ANGPTL4-Sp160 160 ACACCCAGCCAGAACTCGCC (SEQ ID NO: 1429) ANGPTL4-Sp161 161 AGGCGAGTTCTGGCTGGGTC (SEQ ID NO: 1430) ANGPTL4-Sp162 162 GTTCTGGCTGGGTCTGGAGA (SEQ ID NO: 1431) ANGPTL4-Sp163 163 GGAGAAGGTGCATAGCATCA (SEQ ID NO: 1432) ANGPTL4-Sp164 164 GAGAAGGTGCATAGCATCAC (SEQ ID NO: 1433) ANGPTL4-Sp165 165 AGAAGGTGCATAGCATCACG (SEQ ID NO: 1434) ANGPTL4-Sp166 166 GAAGGTGCATAGCATCACGG (SEQ ID NO: 1435) ANGPTL4-Sp167 167 GGGGGACCGCAACAGCCGCC (SEQ ID NO: 1436) ANGPTL4-Sp168 168 GCACGGCCAGGCGGCTGTTG (SEQ ID NO: 1437) ANGPTL4-Sp169 169 GCCGCCTGGCCGTGCAGCTG (SEQ ID NO: 1438) ANGPTL4-Sp170 170 CCGCCTGGCCGTGCAGCTGC (SEQ ID NO: 1439) ANGPTL4-Sp171 171 CCCGCAGCTGCACGGCCAGG (SEQ ID NO: 1440) ANGPTL4-Sp172 172 AGTCCCGCAGCTGCACGGCC (SEQ ID NO: 1441) ANGPTL4-Sp173 173 TGGCCGTGCAGCTGCGGGAC (SEQ ID NO: 1442) ANGPTL4-Sp174 174 GGCCGTGCAGCTGCGGGACT (SEQ ID NO: 1443) ANGPTL4-Sp175 175 ATCCCAGTCCCGCAGCTGCA (SEQ ID NO: 1444) ANGPTL4-Sp176 176 GTGCAGCTGCGGGACTGGGA (SEQ ID NO: 1445) ANGPTL4-Sp177 177 CACGGAGAACTGCAGCCCCT (SEQ ID NO: 1446) ANGPTL4-Sp178 178 GCTGCAGTTCTCCGTGCACC (SEQ ID NO: 1447) ANGPTL4-Sp179 179 CTGCAGTTCTCCGTGCACCT (SEQ ID NO: 1448) ANGPTL4-Sp180 180 CAGTTCTCCGTGCACCTGGG (SEQ ID NO: 1449) ANGPTL4-Sp181 181 CTCCGTGCACCTGGGTGGCG (SEQ ID NO: 1450) ANGPTL4-Sp182 182 GTCCTCGCCACCCAGGTGCA (SEQ ID NO: 1451) ANGPTL4-Sp183 183 GCACCTGGGTGGCGAGGACA (SEQ ID NO: 1452) ANGPTL4-Sp184 184 AGGCCGTGTCCTCGCCACCC (SEQ ID NO: 1453) ANGPTL4-Sp185 185 TGCAGTGAGCTGCAGGCTAT (SEQ ID NO: 1454) ANGPTL4-Sp186 186 CCTGCAGCTCACTGCACCCG (SEQ ID NO: 1455) ANGPTL4-Sp187 187 CCACGGGTGCAGTGAGCTGC (SEQ ID NO: 1456) ANGPTL4-Sp188 188 CAGCTCACTGCACCCGTGGC (SEQ ID NO: 1457) ANGPTL4-Sp189 189 TGCACCCGTGGCCGGCCAGC (SEQ ID NO: 1458) ANGPTL4-Sp190 190 GCACCCGTGGCCGGCCAGCT (SEQ ID NO: 1459) ANGPTL4-Sp191 191 GCGCCCAGCTGGCCGGCCAC (SEQ ID NO: 1460) ANGPTL4-Sp192 192 GGCGCCCAGCTGGCCGGCCA (SEQ ID NO: 1461) ANGPTL4-Sp193 193 GGTGGTGGCGCCCAGCTGGC (SEQ ID NO: 1462) ANGPTL4-Sp194 194 GGACGGTGGTGGCGCCCAGC (SEQ ID NO: 1463) ANGPTL4-Sp195 195 GCCACCACCGTCCCACCCAG (SEQ ID NO: 1464) ANGPTL4-Sp196 196 GCCGCTGGGTGGGACGGTGG (SEQ ID NO: 1465) ANGPTL4-Sp197 197 GAGGCCGCTGGGTGGGACGG (SEQ ID NO: 1466) ANGPTL4-Sp198 198 GGAGAGGCCGCTGGGTGGGA (SEQ ID NO: 1467) ANGPTL4-Sp199 199 GTACGGAGAGGCCGCTGGGT (SEQ ID NO: 1468) ANGPTL4-Sp200 200 GGTACGGAGAGGCCGCTGGG (SEQ ID NO: 1469) ANGPTL4-Sp201 201 AAGGGTACGGAGAGGCCGCT (SEQ ID NO: 1470) ANGPTL4-Sp202 202 GAAGGGTACGGAGAGGCCGC (SEQ ID NO: 1471) ANGPTL4-Sp203 203 AAGTGGAGAAGGGTACGGAG (SEQ ID NO: 1472) ANGPTL4-Sp204 204 TCTCCGTACCCTTCTCCACT (SEQ ID NO: 1473) ANGPTL4-Sp205 205 CTCCGTACCCTTCTCCACTT (SEQ ID NO: 1474) ANGPTL4-Sp206 206 GTCCCAAGTGGAGAAGGGTA (SEQ ID NO: 1475) ANGPTL4-Sp207 207 ACCCTTCTCCACTTGGGACC (SEQ ID NO: 1476) ANGPTL4-Sp208 208 TCCTGGTCCCAAGTGGAGAA (SEQ ID NO: 1477) ANGPTL4-Sp209 209 ATCCTGGTCCCAAGTGGAGA (SEQ ID NO: 1478) ANGPTL4-Sp210 210 GTCGTGATCCTGGTCCCAAG (SEQ ID NO: 1479) ANGPTL4-Sp211 211 ACCAGGATCACGACCTCCGC (SEQ ID NO: 1480) ANGPTL4-Sp212 212 CCAGGATCACGACCTCCGCA (SEQ ID NO: 1481) ANGPTL4-Sp213 213 CCCTGCGGAGGTCGTGATCC (SEQ ID NO: 1482) ANGPTL4-Sp214 214 CGCAGTTCTTGTCCCTGCGG (SEQ ID NO: 1483) ANGPTL4-Sp215 215 TGGCGCAGTTCTTGTCCCTG (SEQ ID NO: 1484) ANGPTL4-Sp216 216 AACTGCGCCAAGAGCCTCTC (SEQ ID NO: 1485) ANGPTL4-Sp217 217 CTGCTCACCAGAGAGGCTCT (SEQ ID NO: 1486) ANGPTL4-Sp218 218 GCAGGGCCTGCTCACCAGAG (SEQ ID NO: 1487) ANGPTL4-Sp219 219 CCCTGACCCCGGCAGGAGGC (SEQ ID NO: 1488) ANGPTL4-Sp220 220 TGACCCCGGCAGGAGGCTGG (SEQ ID NO: 1489) ANGPTL4-Sp221 221 CCGGCAGGAGGCTGGTGGTT (SEQ ID NO: 1490) ANGPTL4-Sp222 222 GTTGAGGTTGGAATGGCTGC (SEQ ID NO: 1491) ANGPTL4-Sp223 223 TGCAGCCATTCCAACCTCAA (SEQ ID NO: 1492) ANGPTL4-Sp224 224 ACTGGCCGTTGAGGTTGGAA (SEQ ID NO: 1493) ANGPTL4-Sp225 225 GAAGTACTGGCCGTTGAGGT (SEQ ID NO: 1494) ANGPTL4-Sp226 226 GACGGAAGTACTGGCCGTTG (SEQ ID NO: 1495) ANGPTL4-Sp227 227 GTGGGATGGAGCGGAAGTAC (SEQ ID NO: 1496) ANGPTL4-Sp228 228 TCCGCTCCATCCCACAGCAG (SEQ ID NO: 1497) ANGPTL4-Sp229 229 GCCGCTGCTGTGGGATGGAG (SEQ ID NO: 1498) ANGPTL4-Sp230 230 CTTCTGCCGCTGCTGTGGGA (SEQ ID NO: 1499) ANGPTL4-Sp231 231 TAAGCTTCTGCCGCTGCTGT (SEQ ID NO: 1500) ANGPTL4-Sp232 232 TTAAGCTTCTGCCGCTGCTG (SEQ ID NO: 1501) ANGPTL4-Sp233 233 GCAGCGGCAGAAGCTTAAGA (SEQ ID NO: 1502) ANGPTL4-Sp234 234 CAGCGGCAGAAGCTTAAGAA (SEQ ID NO: 1503) ANGPTL4-Sp235 235 AGCTTAAGAAGGGAATCTTC (SEQ ID NO: 1504) ANGPTL4-Sp236 236 AGGGAATCTTCTGGAAGACC (SEQ ID NO: 1505) ANGPTL4-Sp237 237 GAATCTTCTGGAAGACCTGG (SEQ ID NO: 1506) ANGPTL4-Sp238 238 AATCTTCTGGAAGACCTGGC (SEQ ID NO: 1507) ANGPTL4-Sp239 239 ATCTTCTGGAAGACCTGGCG (SEQ ID NO: 1508) ANGPTL4-Sp240 240 CGGGTAGTAGCGGCCCCGCC (SEQ ID NO: 1509) ANGPTL4-Sp241 241 GGGCCGCTACTACCCGCTGC (SEQ ID NO: 1510) ANGPTL4-Sp242 242 TGGCCTGCAGCGGGTAGTAG (SEQ ID NO: 1511) ANGPTL4-Sp243 243 ACATGGTGGTGGCCTGCAGC (SEQ ID NO: 1512) ANGPTL4-Sp244 244 AACATGGTGGTGGCCTGCAG (SEQ ID NO: 1513) ANGPTL4-Sp245 245 GGGCTGGATCAACATGGTGG (SEQ ID NO: 1514) ANGPTL4-Sp246 246 CATGGGCTGGATCAACATGG ANGPTL4-Sp247 247 CACCATGTTGATCCAGCCCA (SEQ ID NO: 1516) ANGPTL4-Sp248 248 TGCCATGGGCTGGATCAACA (SEQ ID NO: 1517) ANGPTL4-Sp249 249 GATCCAGCCCATGGCAGCAG (SEQ ID NO: 1518) ANGPTL4-Sp250 250 CTGCCTCTGCTGCCATGGGC (SEQ ID NO: 1519) ANGPTL4-Sp251 251 GAGGCTGCCTCTGCTGCCAT (SEQ ID NO: 1520) ANGPTL4-Sp252 252 GGAGGCTGCCTCTGCTGCCA (SEQ ID NO: 1521) ANGPTL4-Sp252 253 GGCCCAGCCAGGACGCTAGG (SEQ ID NO: 1522)

2. Construction of CjCas9 and sgRNA Plasmids

A sequence encoding human codon-optimized CjCas9 (derived from Campylobacter jejuni subsp. Jejuni NCTC 11168) was synthesized to have a nuclear localization signal (NLS) and an HA epitope at the C-terminus (GeneArt™ Gene Synthesis, Thermo Fisher Scientific), and the synthesized nucleic acid sequence was replicated using a p3s plasmid described in previous research (Cho, S. W., Kim, S., Kim, J. M. & Kim, J. S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nature Biotechnology 31, 230-232 (2013)).

A tracrRNA (transactivating crRNA) sequence and a pre-crRNA (precursor CRISPR RNA) sequence were connected using a GAAA or TGAA linker, thereby producing sgRNA. The sgRNA regulated transcription with an U6 promoter.

3. PAM Characterization Using Cell-Based Reporter Analysis

An AAVS1 target site (AAVS1-TS1) having a variable PAM sequence (5′-NNNNXCAC-3′, 5′-NNNNAXAC-3′, 5′-NNNNACXC-3′, and 5′-NNNNACAX-3′) including a random sequence at the X site was synthesized (Macrogen, Inc.), and the synthesized nucleic acid sequence may be replicated using a surrogate reporter plasmid encoding RFP and GFP.

To determine a suitable PAM sequence, the constructed reporter plasmid (100 ng), and plasmids encoding CjCas9 (225 ng) and sgRNA (675 ng) were co-transfected into HEK293 cells (1×10⁵) using lipofectamine 2000 (Invitrogen). Two days after the transfection, a fractionation of the GFP and RFP-positive cells was measured by flow cytometry (BD Accuri™ C6, BD).

4. Cell Culture and Mutation Analysis

HEK293 (ATCC, CRL-1573) cells and mouse NIH 3T3 (ATCC, CRL-1658) cells were cultured in a Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS).

A sgRNA plasmid (750 ng) and a CjCas9 plasmid (250 ng) were transfected into cells (0.5˜1×10⁵) using lipofectamine 2000 (Invitrogen). 48 hours after the transfection, genome DNA was separated using a DNeasy blood & tissue kit (Qiagen), and an on-target or off-target site was amplified for targeted deep sequencing. Deep sequencing libraries were generated by PCR. TruSeq HT Dual Index primers were used to label respective samples. Mixed libraries were subjected to paired-end sequencing (LAS, Inc.), and indel frequencies were calculated.

5. Construction of AAV Vectors Encoding CjCas9 and sgRNA Sequences

An AAV inverted terminal repeat (ITR)-based vector plasmid containing an sgRNA sequence and a CjCas9 gene having NLS and HA tags at the C-terminus was constructed. sgRNA transcription was induced by an U6 promoter, and CjCas9 expression was regulated by an EFS promoter in C2C12 myoblasts, or by a Spc512 promoter in the TA muscle of C57BL/6 mice.

For retinal delivery, an AAV vector encoding the U6 promoter-induced sgRNA and CjCas9 under the control of the EFS promoter, specific to the Vegfa gene and Hif1a gene, was constructed, wherein CjCas9 had eGFP linked to the C-terminus using a self-cleaved T2A peptide.

6. Production, Purification and Characterization of AAV Vector

To produce the AAV vector, a pseudotype of AAVDJ or AAV9 capsids was used. The HEK293T cells were transfected with pAAV-ITR-CjCas9-sgRNA, pAAVED2/9, and a helper plasmid. The HEK293T cells were cultured in a 2% FBS-containing DMEM. Recombinant AAV vector stocks were produced using PEI coprecipitation by mixing polyplus-transfection (PElpro), triple-transfection and the plasmid in a molar ratio of 1:1:1 in the HEK293T cells. After 72 hours of culturing, the cells were lysed, and particles were isolated and purified with iodixanol (Sigma-Aldrich) by step-gradient ultracentrifugation. The number of vector genomes was quantified through quantitative PCR.

7. AAV Transduction in Mouse Myoblasts

Mouse myoblasts were infected with various viral amounts of AAVDJ-CjCas9 (multiplicity of infection (MOI): 1, 5, 10, 50, and 100 (determined by quantitative PCR)), and cultured in 2% FBS-containing DMEM. For the target deep sequencing, cells were obtained at different points of time. MOI 1 is considered as infection by one virus particle among a total of 100 virus particles determined by quantitative PCR.

8. Animals

Management, use and treatment of all animals used in this research were performed under the guidance provided by the Seoul National University Animal Care and Use Committee, ophthalmology and security research, and the strict agreement according to the ARVO statement on animal use in veterinary medicine. In this research, specific male pathogen free (SPF)-6-week old C57BL/6J mice were used. The mice were maintained in a 12-hour light/dark cycle.

Diabetic model mice were induced by injecting streptozotocin (STZ, Sigma-Aldrich, St. Louis, Mo., USA) intraperitonially once. As a control, a citrate buffer was injected. 4 days after the STZ injection, when a blood glucose level of the mouse was 300 mg/dl or more, diabetes was considered to be induced.

9. Injection of AAV into Vitreous Body

A mixture of tiletamine and zolazepam in a ratio of 1:1 (2.25 mg/kg (body weight) each), and 0.7 mg/kg (body weight) of xylazine hydrochloride was injected into a vitreous body of a 6-week old mouse for anesthetization. 2 μl (2×1010 viral genome) of AAV9-CjCas9 was injected into the vitreous body using a Nanofil syringe (World Precision Instruments Inc.) having a 33G blunt needle under a surgical microscope (Leica Microsystems Ltd.).

In the case of diabetic mouse models, the mice were injected with STZ to induce diabetes, anesthetized after 1 or 7 weeks, and 2 μl of the mixture containing 0.8×108 vg/μl of CjCas9:Vegfa or 1.5×109 vg/μl of Rosa26 was injected in the vitreous body.

10. Immunofluorescence Staining and Imaging of Retinal Tissue

42 days after the injection, the sample was fixed with formalin and embedded in paraffin (n=4). A sample obtained by cross-section of the sample-embedded paraffin was immunostained with an anti-HA antibody (Roche, 3F10, 1:1000), an anti-opsin antibody (Millipore, AB5405, 1:1000), and an Alexa Fluor 488 or 594 antibody (Thermo Fisher Scientific, 1:500). An opsin-positive site was detected in RPE cells expressing HA-tagged CjCas9 using Image J software (1.47v, NIH). The distribution of CjCas9 and eGFP was visualized on RPE flat-mounts using a confocal microscope (LSM 710, Carl Zeiss).

11. Extraction of Genome DNA

To extract DNA from RPE and the retina, after obtaining images of RPE and the retina flat-mounts, the tissue samples were washed with PBS. RPE cells were separated from the choroid/sclera by vortexing for 30 seconds in a lysis buffer (NucleoSpin Tissue, Macherey-Nagel). Genome DNA was analyzed to identify complete isolation of the RPE cells from remaining choroid/sclera tissue. The genome DNA was analyzed by target deep sequencing.

12. Mouse Vegfa ELISA

42 days after the injection, a total RPE mixture was isolated from neural retina tissue, and two kinds of tissue were frozen for subsequent analysis. The sample tissue was lysed in 120 μl of a cell lysis buffer (CST #9803), and the amount of a Vegfa protein was measured using a mouse VEGF Quantikine ELISA kit (MMVOO, R&D Systems).

13. Laser-Induced CNV Model

After anesthetization of a mouse, eye drops containing 0.5% phenylephrine and 0.5% tropicamide were injected to dilate a pupil. Laser photocoagulation was performed using an indirect head set delivery system (Iridex) and a laser system (Ilooda). Parameters of the laser are a wavelength of 532 nm, a spot size of 200 μm, a power of 800 mW and exposure time of 70 ms. A laser burn was induced in the proximity of the optical nerve three to four times. Burns in which bubbles are generated without bleeding of the vitreous body were only used for the research. After 7 days, an eyeball was fixed with 4% paraformaldehyde at room temperature for one hour. An RPE mixture (RPE/choroid/sclera) was immunostained overnight at 4° C. using isolectin-B4 (Thermo Fisher Scientific, cat. no. 121413, 1:100) and an anti-GFP antibody (Abcam, ab6556, 1:100). The RPE mixture was flat-mounted, and visualized using a fluorescent microscope (Eclipse 90i, Nikon) or a confocal microscope (LSM 710, Carl Zeiss) at a 100× magnification. A CNV site was detected using Image J software (1.47v, NIH). An average of three to four CNV sites per eyeball was analyzed. Each group consists of 17 to 18 eyeballs.

14. Quantitative and Qualitative Analyses for Rupturing of Retinal Vessels

To detect vascular leakage, STZ-induced diabetic mouse models were used. 200 μl of an Evans blue dye (20 mg/ml) dissolved in PBS was intravenously injected into an anesthetized mouse. Two hours after perfusion, an eyeball was extracted to be fixed with 4% paraformaldehyde for 1 hour. The retina was excised in 2×PBS and flat-mounted, and images of the retina were obtained at 40× and 100× magnifications using a fluorescent microscope (Eclipse 90i, Nikon).

To quantitatively analyze the vascular leakage, representative four sites of the vascular leakage in the mid-peripheral retina (0.5 μm×0.5 μm) of each mouse were selected. The mid-peripheral retina was designated as the middle 1/3 of the retina from the optic nerve head to the ciliary body, images were modulated according to color threshold values based on the automatic isodata algorithm using the Image J software (1.47v, NIH), and regions of interest containing the Evans blue dye were marked in red. Afterward, the regions marked in red were detected. The data were normalized with data of control mice, and represented as vascular leakage (%).

15. Data Analysis

To previously determine a sample size in vitro or in vivo, a statistical method was not used. For statistical analysis, one-way ANOVA and Tukey post-hoc tests were used.

Example 1. Confirmation of CjCas9 Expression Through AAV in Mouse Retinal Tissue

Since CjCas9 consisting of 984 amino acids has a considerably smaller size (2.95 kbp) than SpCas9, both CjCas9 gene and sgRNA are able to be packaged in one AAV vector. Therefore, in this example, to confirm the possibility of gene manipulation using CjCas9 as a method for treating AMD, CjCas9-expressing AAV was used.

To confirm the expression of CjCas9 through AAV in tissue such as the retina in a mouse, under the control of U6 promoter-induced sgRNA and an EFS promoter specific to a choroidal neovascularization (CNV)-associated Vegfa gene and an Hif1a gene, a CjCas9-coding AAV9 vector was constructed, and here, CjCas9 was linked to eGFP at the C-terminus using a self-cleaved T2A peptide (FIGS. 1 and 2). The constructed virus was injected into an eyeball through injection into the vitreous body, and after 6 weeks, CjCas9 expression in the eyeball was confirmed, an indel frequency was measured using target deep sequencing, and the amount of Vegfa protein was measured through ELISA (FIG. 4).

The expression of the CjCas9-linked eGFP was confirmed in retinal pigment epithelial (RPE) cells (FIG. 8).

In addition, indels induced by CjCas9 were observed at Rosa26, Vegfa, and Hif1a target sites of the RPE cells, indel frequencies of 14±5%, 22±3%, and 31±2% were confirmed, respectively (FIG. 3). In addition, noticeable off-target indels were not induced by Vegfa- or Hif1a-specific sgRNA in the cells (FIG. 5).

As expected, the expression level of the Vegfa protein was decreased in AAV-treated RPE cells encoding Vegfa-specific CjCas9 (AAV-CjCas9: Vegfa), but was not when Hif1a- or Rosa26-specific CjCas9 (AAV-CjCas9: Hif1a or Rosa26)-coding AAV was treated (FIG. 4). Particularly, since a Hif1a protein is degraded under a normoxia condition, the expression level of the protein was not able to be measured.

Example 2. Effect of Vegfa- or Hif1a-Specific AAV-CjCas9 Using CNV-Induced Mouse

To induce CNV, an eyeball was injected with AAV, and after 6 weeks, subjected to laser treatment, followed by detecting the CNV sites one week after the laser treatment (FIG. 6). When AAV-CjCas9: Vegfa and AAV-CjCas9: Hif1a were injected onto respective eyeballs, it was confirmed that, compared with an AAV-free negative control, CNV sites were decreased 24±4% and 20±4%, respectively (FIGS. 6 and 7). In the case of another negative control, that is, Rosa26-specific CjCas9, no therapeutic effect was confirmed, either.

Example 3. Effect of AAV-CjCas9 for AMD Treatment

Since cone dysfunction is caused by conditional knockout of a Vegfa gene in the mouse RPE cells, the cause of side effects by the AAV-induced gene knockout in the RPE cells was investigated. To this end, the size of a cone function-related opsin-positive site in the retina was measured. As a result, the Vegfa-specific CjCas9 was decreased in size by approximately 30±10% the AAV-free control (FIGS. 9 and 10). However, as expected, the Hif1a- or Rosa26-specific CjCas9 did not induce cone dysfunction. Such a result may suggest that Hif1a inactivation for treatment of AMD inhibits neovascularization and does not cause cone dysfunction.

HIF1A is a hypoxia-inducible transcription factor, and serves to activate VEGFA transcription. Unlike VEGFA, which is a primary therapeutic target for AMD treatment and a secretory protein, HIF1A may not be considered as a drug target, and generally, a transcription factor such as HIF1A may not be directly targeted by an antibody or aptamer, or small molecules. In this research, as the Hif1a gene was effectively inactivated in the RPE cells using CjCas9 targeting the Hif1a gene on the eyeball of a mouse, it was confirmed that the CNV sites were reduced in the AMD mouse models (FIGS. 9 and 10).

Example 4. Effect of AAV-CjCas9 for Treatment of Retinal Disease

For extended application to retinal diseases such as diabetic retinopathy (DR), retinopathy of prematurity, etc., the constructed virus was injected into the eyeball through injection into the vitreous body, and 6 weeks later, the in vivo genome editing effect caused by the indel frequency in retinal tissue was observed by target deep sequencing, followed by confirming an expression level of the Vegfa protein through ELISA (FIG. 2). In the retinal tissue, the indels induced by CjCas9 were observed at the Rosa26, Vegfa, and Hif1a target sites in the retinal cells, indel frequencies of 44±8%, 20±2%, and 58±5% were confirmed, respectively (FIG. 11). As expected, an expression level of the Vegfa protein was decreased in retinal cells when treated with AAV encoding Vegfa- or Hif1a-specific CjCas9 (AAV-CjCas9: Vegfa or Hif1a), but did not when AAV encoding Rosa26-specific CjCas9 (AAV-CjCas9: Rosa26) was treated (FIG. 12).

Example 5. Effect of AAV-CjCas9 for Treatment of Diabetic Retinopathy

Diabetic retinopathy is characterized by the symptoms of vascular leakage and blood leakage. Therefore, in this example, the vascular leakage or blood leakage symptom was confirmed using diabetes-induced mice using STZ, and an improvement or treatment effect caused by CjCas9 was confirmed. As a result, it was confirmed that the blood leakage caused by vascular leakage and rupturing was decreased in the Vegfa-specific CjCas9 (AAV-CjCas9: Vegfa)-injected retina. Compared with Rosa26-specific CjCas9 (AAV-CjCas9: Rosa26)-injected mice, vascular leakage and blood leakage in Vegfa-specific CjCas9 (AAV-CjCas9: Vegfa)-injected mice were decreased and thus recovered to a level similar as in normal mice of the same age. Such a result was similarly shown in both of an experiment in which STZ was injected, AAV-CjCas9 was injected after 7 weeks and observation was performed after 6 weeks (FIG. 13), and an experiment in which STZ was injected, AAV-CjCas9 was injected after 14 weeks and observation was performed after 7 weeks (FIGS. 14 and 15). According to the above results, it was confirmed that Vegfa-specific CjCas9 (AAV-CjCas9: Vegfa) has an effect of reducing vascular leakage and blood leakage, and thus it can be expected that the Vegfa-specific CjCas9 (AAV-CjCas9: Vegfa) can effectively treat diabetic retinopathy.

Example 6. Screening of Target Site of Human Neovascularization-Associated Factor

To extend the application of the previously-described example, in addition to human VEGFA (FIG. 16) and human HIF1A (FIG. 17), human ANGPT2 (FIG. 19), human EPAS1 (FIG. 20) and human ANGPTL4 (FIG. 21) were selected as potential targets of genome editing for AMD and DR treatments to screen the target site of each gene capable of being effectively edited in human cells using a CjCas9 system. Particularly, the CjCas9 target site of the mouse Hif1a gene was completely conserved in a human or a different mammal (FIG. 18). Additionally, the high editing efficiency of the conserved target site was observed in human cells (FIG. 17, sgRNA #7). Therefore, it is expected that AAV for Hif1a suggested in this research or a mutant thereof is able to be used in treatment of a future human patient.

INDUSTRIAL APPLICABILITY

An artificially manipulated neovascularization-associated factor and a neovascularization system artificially modified in function thereby can be effectively used in treatment of an angiogenic disease, for example, an angiogenesis-associated ocular disease.

Efficiency of the neovascularization system can be improved by regulating characteristics such as survival, proliferation, persistency, cytotoxicity, and cytokine-release of various neovascularization-associated factors.

Original Claims

1. An artificially manipulated neovascularization-associated factor, which is selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, which has a modification in a nucleic acid sequence. 2. The artificially manipulated neovascularization-associated factor of paragraph 1, wherein the modification in the nucleic acid sequence is artificially caused by a guide nucleic acid-editor protein complex. 3. The artificially manipulated neovascularization-associated factor of paragraph 1, wherein the neovascularization-associated factor is one or more selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene. 4. The artificially manipulated neovascularization-associated factor of paragraph 1, wherein the gene is a neovascularization-associated factor artificially manipulated by a guide nucleic acid-editor protein complex, wherein the neovascularization-associated factor artificially manipulated includes one or more modifications of nucleic acids which is at least one of a deletion or insertion of one or more nucleotides, a substitution with one or more nucleotides different from a wild-type gene, and an insertion of one or more foreign nucleotide, in a proto-spacer-adjacent motif (PAM) sequence in a nucleic acid sequence constituting the neovascularization-associated factor or in a continuous 1 bp to 50 bp the base sequence region adjacent to the 5′ end and/or 3′ end thereof, or a chemical modification of one or more nucleotides in a nucleic acid sequence constituting the neovascularization-associated factor. 5. The artificially manipulated neovascularization-associated factor of paragraph 1, wherein the modification of nucleic acids occurs in a promoter region of the gene. 6. The artificially manipulated neovascularization-associated factor of paragraph 1, wherein the modification of nucleic acids occurs in an exon region of the gene. 7. The artificially manipulated neovascularization-associated factor of paragraph 1, wherein the modification of nucleic acids occurs in an intron region of the gene. 8. The artificially manipulated neovascularization-associated factor of paragraph 1, wherein the modification of nucleic acids occurs in an enhancer region of the gene. 9. The artificially manipulated neovascularization-associated factor of paragraph 4, wherein the PAM sequence is, one or more of the following sequences (described in the 5′ to 3′ direction): NGG (N is A, T, C or G); NNNNRYAC (each N is independently A, T, C or G, R is A or G, and Y is C or T); NNAGAAW (each N is independently A, T, C or G, and W is A or T); NNNNGATT (each N is independently A, T, C or G); NNGRR(T) (each N is independently A, T, C or G, R is A or G); and TTN (N is A, T, C or G). 10. The artificially manipulated neovascularization-associated factor of paragraph 2, wherein the editor protein includes one or more selected from the group consisting of a Streptococcus pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived Cas9 protein, a Streptococcus aureus-derived Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a Cpf1 protein. 11. A guide nucleic acid, which is capable of forming complementary bonds with respect to the target sequences of SEQ ID NOs: 1 to 79 in the nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, respectively. 12. The guide nucleic acid of paragraph 11, which includes one or more guide nucleic acids selected from the group consisting of: guide nucleic acids capable of forming complementary bonds with respect to the target sequences of SEQ ID NOs: 3, 4, 7, 9, 10 and 11 in the nucleic acid sequence of the VEGFA gene, respectively; guide nucleic acids capable of forming complementary bonds with respect to the target sequences of SEQ ID NOs: 14, 18, 19, 20, 26, 29 and 31 in the nucleic acid sequence of the HIF1A gene, respectively; guide nucleic acids capable of forming complementary bonds with respect to the target sequences of SEQ ID NOs: 33, 34, 37, 38, 39 and 43 in the nucleic acid sequence of the ANGPT2 gene, respectively; guide nucleic acids capable of forming complementary bonds with respect to the target sequences of SEQ ID NOs: 47, 48, 49, 50, 53, 54 and 55 in the nucleic acid sequence of the EPAS1 gene, respectively; and guide nucleic acids capable of forming complementary bonds with respect to the target sequences of SEQ ID NOs: 64, 66, 67, 73, 76 and 79 in the nucleic acid sequence of the ANGPTL4 gene, respectively. 13. The guide nucleic acid of paragraph 11, wherein the guide nucleic acid is nucleotide molecule of 18 to 23 bp. 14. A composition for gene manipulation, comprising: a guide nucleic acid capable of forming a complementary bond with respect to the target sequences of SEQ ID NOs: 1 to 79 in nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, respectively, or a nucleic acid sequence encoding the same; and an editor protein or a nucleic acid sequence encoding the same. 15. The composition for gene manipulation of paragraph 14, wherein the editor protein includes one or more proteins selected from the group consisting of a Streptococcus pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived Cas9 protein, a Streptococcus aureus-derived Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a Cpf1 protein. 16. The composition for gene manipulation of paragraph 14, wherein the gene manipulation includes one or more modifications of nucleic acids which is at least one of a deletion or insertion of one or more nucleotides, a substitution with one or more nucleotides different from a wild-type gene, and an insertion of one or more foreign nucleotide, in a proto-spacer-adjacent motif (PAM) sequence in a nucleic acid sequence constituting the neovascularization-associated factor or in a continuous 1 bp to 50 bp the base sequence region adjacent to the 5′ end and/or 3′ end thereof, or a chemical modification of one or more nucleotides in a nucleic acid sequence constituting the neovascularization-associated factor. 17. The composition for gene manipulation of paragraph 16, wherein the PAM sequence includes one or more of the following sequences (described in the 5′ to 3′ direction): NGG (N is A, T, C or G); NNNNRYAC (each N is independently A, T, C or G, R is A or G, and Y is C or T); NNAGAAW (each N is independently A, T, C or G, and W is A or T); NNNNGATT (each N is independently A, T, C or G); NNGRR(T) (each N is independently A, T, C or G, R is A or G); and TTN (N is A, T, C or G). 18. The composition for gene manipulation of paragraph 14, wherein the composition for gene manipulation is formed in a viral vector system. 19. The composition for gene manipulation of paragraph 18, wherein the viral vector includes one or more selected from a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia virus, a poxvirus and a herpes simplex virus. 20. A method for providing information on a sequence of an artificially manipulatable target site in a subject by analyzing sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene. 21. A method for constructing a library using the information provided by the method of claim 20. 22. A kit for gene manipulation, comprising: (a) a guide nucleic acid capable of forming complementary bonds with respect to each of the target sequences of SEQ ID NOs: 1 to 79, in the nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, respectively, or a nucleic acid sequence encoding the same; and (b) an editor protein including one or more proteins selected from the group consisting of a Streptococcus pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived Cas9 protein, a Streptococcus aureus-derived Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a Cpf1 protein, respectively, or a nucleic acid sequence encoding the same. 23. A composition for treating an angiovascular disorder, comprising: a guide nucleic acid capable of forming complementary bonds with respect to each of one or more target sequences in the nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, respectively, or a nucleic acid sequence encoding the same; and an editor protein or a nucleic acid sequence encoding the same. 24. The composition for treating of paragraph 23, wherein the target sequence includes one or more of target sequences of SEQ ID NOs: 1 to 79. 25. The composition for treating of paragraph 23, wherein the editor protein is a Campylobacter jejuni-derived Cas9 protein. 26. The composition for treating of paragraph 23, wherein the angiovascular disorder is ischemic retinopathy or retinopathy of prematurity.

SEQUENCE LISTING

This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted herewith as the sequence listing text file. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e). 

What is claimed is:
 1. A composition for gene manipulation, comprising: a guide nucleic acid capable of targeting at least one of the target sequence selected from SEQ ID NOs: 1 to 1522 in nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, or a nucleic acid sequence encoding the same; and an editor protein or a nucleic acid sequence encoding the same.
 2. The composition for gene manipulation of claim 1, wherein the editor protein includes one or more proteins selected from the group consisting of a Streptococcus pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived Cas9 protein, a Streptocuccus aureus-derived Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a Cpf1 protein.
 3. The composition for gene manipulation of claim 2, wherein the editor protein is a Streptococcus pyogenes-derived Cas9 protein or Campylobacter jejuni-derived Cas9 protein.
 4. The composition for gene manipulation of claim 1, wherein the gene manipulation includes one or more modifications of nucleic acids which is at least one of a deletion or insertion of one or more nucleotides, a substitution with one or more nucleotides different from a wild-type gene, and an insertion of one or more foreign nucleotide, in a proto-spacer-adjacent motif (PAM) sequence in a nucleic acid sequence constituting the neovascularization-associated factor or in a continuous 1 bp to 50 bp the base sequence region adjacent to the 5′ end and/or 3′ end thereof, or a chemical modification of one or more nucleotides in a nucleic acid sequence constituting the neovascularization-associated factor.
 5. The composition for gene manipulation of claim 4, wherein the PAM sequence includes one or more of the following sequences (described in the 5′ to 3′ direction): NGG (N is A, T, C or G); NNNNRYAC (each N is independently A, T, C or G, R is A or G, and Y is C or T); NNAGAAW (each N is independently A, T, C or G, and W is A or T); NNNNGATT (each N is independently A, T, C or G); NNGRR(T) (each N is independently A, T, C or G, R is A or G, and Y is C or T); and TTN (N is A, T, C or G).
 6. The composition for gene manipulation of claim 1, wherein the composition for gene manipulation is formed in a viral vector system.
 7. The composition for gene manipulation of claim 6, wherein the viral vector includes one or more selected from a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia virus, a poxvirus and a herpes simplex virus.
 8. The composition for gene manipulation of claim 1, wherein the composition is formed in a ribonucleoprotein which is a complex of a guide nucleic acid and an editor protein, and wherein the guide nucleic acid is guide RNA.
 9. The composition for gene manipulation of claim 1, wherein the composition is used for treating an angiovascular disorder.
 10. The composition for treating of claim 9, wherein the angiovascular disorder is ischemic retinopathy or retinopathy of prematurity.
 11. A guide nucleic acid, which is capable of targeting at least one of group consisting of target sequences of SEQ ID NOs: 1 to 1522 for artificially manipulating one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, wherein the target sequences of SEQ ID NOs: 1 to 1522 are in the nucleic acid sequences of the genes, and wherein the guide nucleic acid is complexed with an editor protein for artificially manipulating the genes.
 12. The guide nucleic acid of claim 11, which includes one or more guide nucleic acids selected from the group consisting of: guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 3, 4, 7, 9, 10 and 11 in the nucleic acid sequence of the VEGFA gene; guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 14, 18, 19, 20, 26, 29 and 31 in the nucleic acid sequence of the HIF1A gene; guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 33, 34, 37, 38, 39 and 43 in the nucleic acid sequence of the ANGPT2 gene; guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 47, 48, 49, 50, 53, 54 and 55 in the nucleic acid sequence of the EPAS1 gene; and guide nucleic acids capable of targeting at least one of the target sequence selected from SEQ ID NOs: 64, 66, 67, 73, 76 and 79 in the nucleic acid sequence of the ANGPTL4 gene.
 13. The guide nucleic acid of claim 11, wherein the guide nucleic acid is a nucleotide of 18 to 23 bp.
 14. A method for treating an angiovascular disorder, comprising: introducing (administering) a composition to a subject, wherein the composition comprising: a guide nucleic acid capable of targeting at least one of the target sequence selected from SEQ ID NOs: 1 to 1522 in nucleic acid sequences of one or more genes selected from the group consisting of a VEGFA gene, an HIF1A gene, an ANGPT2 gene, an EPAS1 gene and an ANGPTL4 gene, or a nucleic acid sequence encoding the same; and an editor protein or a nucleic acid sequence encoding the same.
 15. The method of claim 14, wherein the angiovascular disorder is ischemic retinopathy or retinopathy of prematurity.
 16. The method of claim 14, wherein the editor protein includes one or more proteins selected from the group consisting of a Streptococcus pyogenes-derived Cas9 protein, a Campylobacter jejuni-derived Cas9 protein, a Streptococcus thermophilus-derived Cas9 protein, a Streptocuccus aureus-derived Cas9 protein, a Neisseria meningitidis-derived Cas9 protein, and a Cpf1 protein.
 17. The method of claim 14, wherein the introducing is conducted through eyeball.
 18. The method of claim 14, wherein the composition is formed in a viral vector system.
 19. The method of claim 18, wherein the viral vector includes one or more selected from a retrovirus, a lentivirus, an adenovirus, adeno-associated virus (AAV), vaccinia virus, a poxvirus and a herpes simplex virus. 